Antivirally active heterocyclic azahexane derivatives

ABSTRACT

There are described compounds of formula I*,                    
     wherein 
     R 1  is lower alkoxycarbonyl, 
     R 2  is secondary or tertiary lower alkyl or lower alkylthio-lower alkyl, 
     R 3  is phenyl that is unsubstituted or substituted by one or more lower alkoxy radicals, or C 4 -C 8 cycloalkyl, 
     R 4  is phenyl or cyclohexyl each substituted in the 4-position by unsaturated heterocyclyl that is bonded by way of a ring carbon atom, has from 5 to 8 ring atoms, contains from 1 to 4 hetero atoms selected from nitrogen, oxygen, sulfur, suffinyl (—SO—) and sulfonyl (—SO 2 —) and is unsubstituted or substituted by lower alkyl or by phenyl-lower alkyl, 
     R 5 , independently of R 2 , has one of the meanings mentioned for R 2 , and 
     R 6 , independently of R 1 , is lower alkoxycarbonyl, 
     or salts thereof, provided that at least one salt-forming group is present. 
     The compounds are inhibitors of retroviral aspartate protease and can be used, for example, in the treatment of AIDS. They exhibit outstanding pharmacodynamic properties.

CROSS-REFERENCE

This application is a divisional of application Ser. No. 09/108,681filed Jul. 1, 1998 now U.S. Pat. No. 6,110,946.

The invention relates to heterocyclic azahexane derivatives that can beemployed as substrate isosteres of retroviral aspartate proteases, tosalts thereof, to processes for the preparation of those compounds andtheir salts, to pharmaceutical compositions comprising those compoundsor their salts, and to the use of those compounds or their salts (aloneor in combination with other antiretrovirally active compounds) in thetherapeutic or diagnostic treatment of the human or animal body or inthe preparation of pharmaceutical compositions.

BACKGROUND OF THE INVENTION

According to WHO estimates there are clearly more than 20 million peopleinfected by the “Human Immuno Deficiency Virus”, HIV-1 or HIV-2. Withvery few exceptions, in infected subjects the disease results, by way ofpreliminary stages, such as ARDS, in a manifest disease of the immunesystem which is known as “Acquired Immunodeficiency Syndrome” or AIDS.In the overwhelming number of cases the disease sooner or later leads tothe death of the infected patients.

Hitherto, the treatment of retroviral diseases, such as AIDS, hasinvolved principally the use of inhibitors of reverse transcriptase, anenzyme effective in the conversion of retroviral RNA into DNA, such as3′-azido-3′-deoxythymidine (AZT) or dideoxyinosine (DDI), and alsotrisodium phosphonoformate, ammonium-21-tungstenato-9-antimonate,1-β-D-ribofuran-oxyl-1,2,4-triazole-3-carboxamide and dideoxycytidineand also adriamycin. Attempts have also been made to introduce into thebody, for example in the form of a recombinant molecule or moleculefragment, the T4-cell receptor which is present on certain cells of thedefence system of the human body and is responsible for the anchoringand introduction of infectious virus particles into those cells and thusfor their infection, the objective being that binding sites for thevirus will be blocked so that the virions will no longer be able to bindto the cells. Compounds that prevent the virus penetrating the cellmembrane in some other way, such as polymannoacetate, are also used.

The first inhibitor of so-called retroviral aspartate protease to beapproved for combatting the infection was saquinavir,[N-tert-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-2-quinolyl-carbonyl-L-asparaginyl]amino]butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide(Ro 31-8959)]. Since then others have followed (indinavir (Merck) andritonavir (Abbott)).

Also under development are a number of further inhibitors of retroviralaspartate protease, an enzyme the function of which can be characterisedas follows:

In the AIDS viruses, HIV-1 and HIV-2, and other retroviruses, forexample corresponding viruses in cats (FIV) and apes (SIV), theproteolytic maturation of, for example, the core proteins of the virusis brought about by an aspartate protease, such as HIV-protease. Withoutthat proteolytic maturation, infectious virus particles cannot beformed. Owing to the central role of the said aspartate proteases, suchas HIV-1- or HIV-2-protease, in the maturation of viruses and on thebasis of experimental results, for example on infected cell cultures, ithas become plausible that effective suppression of the maturation stepbrought about by that protease will suppress the assembly of maturevirions in vivo. Inhibitors of that protease can therefore be usedtherapeutically.

The aim of the present invention is to provide a novel type of compoundthat is equipped, especially, with a high degree of inhibitory activityagainst virus replication in cells, high anti-viral activity againstnumerous virus strains, including those which are resistant to knowncompounds, such as saquinavir, ritonavir and indinavir, and especiallyadvantageous pharmacological properties, for example goodpharmacokinetics, such as high bioavailability and high blood levels,and/or high selectivity.

FULL DESCRIPTION OF THE INVENTION

The azahexane derivatives according to the invention are compounds offormula I*,

especially of formula I,

wherein

R₁ is lower alkoxycarbonyl,

R₂ is secondary or tertiary lower alkyl or lower alkylthio-lower alkyl,

R₃ is phenyl that is unsubstituted or substituted by one or more loweralkoxy radicals, or C₄-C₈cycloalkyl,

R₄ is phenyl or cyclohexyl each substituted in the 4-position byunsaturated heterocyclyl that is bonded by way of a ring carbon atom,has from 5 to 8 ring atoms, contains from 1 to 4 hetero atoms selectedfrom nitrogen, oxygen, sulfur, sulfinyl (—SO—) and sulfonyl (—SO₂—) andis unsubstituted or substituted by lower alkyl or by phenyl-lower alkyl,

R₅, independently of R₂, has one of the meanings mentioned for R₂, and

R₆, independently of R₁, is lower alkoxycarbonyl,

or a salt thereof, provided that at least one salt-forming group ispresent.

Those compounds exhibit unexpectedly good and surprisingly positivepharmacological properties, as indicated in detail below, and arerelatively simple to synthesise.

Unless indicated to the contrary, the general terms used hereinabove andhereinbelow preferably have the following meanings within the scope ofthis disclosure:

The term “lower” indicates a radical having up to and including amaximum of 7 carbon atoms, preferably up to and including a maximum of 4carbon atoms, the radicals in question being unbranched or branched oneor more times.

Lower alkyl and C₁-C₄alkyl are especially tert-butyl, sec-butyl,isobutyl, n-butyl, isopropyl, n-propyl, ethyl and methyl.

Any reference to compounds, salts and the like in the plural alsoincludes a compound, a salt and the like.

Any asymmetric carbon atoms present, for example the carbon atoms bondedto the radicals R₂ and R₅, may be in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration, the(S)-configuration being especially preferred in the case of the carbonatoms carrying the radical R₂ and/or R₅ in compounds of formula I.Accordingly, the compounds in question may be in the form of isomericmixtures or in the form of pure isomers, preferably in the form ofenantiomerically pure diastereoisomers.

Lower alkoxycarbonyl is preferably C₁-C₄alkoxycarbonyl wherein the alkylradical may be branched or unbranched, and is especially ethoxycarbonylor methoxycarbonyl.

Secondary or tertiary lower alkyl is especially sec-butyl, tert-butyl orisopropyl.

Lower alkylthio-lower alkyl is especially methylthiomethyl.

Phenyl that is unsubstituted or substituted by one or more lower alkoxyradicals is especially phenyl that is unsubstituted or substituted byfrom one to three lower alkoxy radicals, especially methoxy. In the casewhen there are three methoxy substituents, these are especially in the2,3,4-positions of the phenyl ring and in the case when there is onemethoxy substituent, that substituent is especially in the 2-, 3- or,more especially, in the 4-position. Unsubstituted phenyl is preferred.

C₄-C₈cycloalkyl is especially cyclopentyl or, more especially,cyclohexyl.

As R₃ phenyl is preferred to cyclohexyl.

In phenyl or cyclohexyl substituted in the 4-position by unsaturatedheterocyclyl that is bonded by way of a ring carbon atom, has from 5 to8 ring atoms, contains from 1 to 4 hetero atoms selected from nitrogen,oxygen, sulfur, sulfinyl (—SO—) and sulfonyl (—SO₂—) and isunsubstituted or substituted by lower alkyl or by phenyl-lower alkyl,the corresponding heterocyclyl has especially the following meanings:

Unsaturated heterocyclyl that is bonded by way of a ring carbon atom,has from 5 to 8 ring atoms, contains from 1 to 4 hetero atoms selectedfrom nitrogen, oxygen, sulfur, sulfinyl (—SO—) and sulfonyl (—SO₂—) andis unsubstituted or substituted by lower alkyl, especially by methyl, orby phenyl-lower alkyl wherein the lower alkyl radical is unbranched orbranched, especially by 1-methyl-1-phenylethyl, is especially one of thefollowing radicals bonded by way of a ring carbon atom: thienyl(=thiophenyl); oxazolyl; thiazolyl; imidazolyl; 1,4-thiazinyl; triazolylthat is unsubstituted or, especially, substituted by1-methyl-1-phenyl-ethyl or preferably by tert-butyl or especially bymethyl, such as 1-, 2- or 4-(methyl or tert-butyl)-triazol-3-yl;tetrazolyl that is unsubstituted or, especially, substituted by1-methyl-1-phenyl-ethyl or preferably by lower alkyl, such as bytert-butyl or especially by methyl, such as 2H-tetrazol-5-yl substitutedby 1-methyl-1-phenyl-ethyl or preferably by lower alkyl, such as bytert-butyl or especially by methyl, or 1H-tetrazol-5-yl substituted bytert-butyl or especially by methyl; pyridinyl; pyrazinyl; andpyrimidinyl; more especially, 2- or 3-thienyl (=thiophen-2-yl orthiophen-3-yl); thiazol-5-yl; thiazol-2-yl; 2H-tetrazol-5-yl that isunsubstituted or, especially, substituted in the 2-position by1-methyl-1-phenyl-ethyl or preferably by tert-butyl or especially bymethyl; 1H-tetrazol-5-yl substituted in the 1-position by methyl;pyridin-2-yl; pyridin-3-yl; pyridin-4-yl; or pyrazin-2-yl.

R₄ is preferably phenyl substituted in the 4-position by unsaturatedheterocyclyl that is bonded by way of a ring carbon atom, has from 5 to8 ring atoms, contains from 1 to 4 hetero atoms selected from nitrogen,oxygen, sulfur, sulfinyl (—SO—) and sulfonyl (—SO₂—) and isunsubstituted or substituted by lower alkyl or by phenyl-lower alkyl,wherein heterocyclyl preferably has the meanings defined above as beingpreferred.

The compounds of formula I preferably have the formula Ia,

wherein the radicals are as defined.

Salts are especially the pharmaceutically acceptable salts of compoundsof formula I.

Such salts are formed, for example, by compounds of formula I having abasic R₄—CH₂— carrying nitrogen atom as acid addition salts, preferablywith inorganic acids, for example hydrohalic acid, such as hydrochloricacid, sulfuric acid or phosphoric acid, or with strong organic sulfonic,sulfo or phosphoric acids or N-substituted sulfamic acids (preferably:pKa<1). Other salts may be present when basic heterocyclyl radicals,such as pyridyl, are present in R₄. Those salts includes especially acidaddition salts with organic or inorganic acids, especially thepharmaceutically acceptable salts. Suitable inorganic acids are, forexample, hydrchalic acids, such as hydrochloric acid, sulfuric acid andphosphoric acid. Suitable organic acids are, for example, carboxylic,phosphonic, sulfonic or sulfamic acids, for example acetic acid,propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolicacid, lactic acid, 2-hydroxybutyric acid, gluconic acid,glucosemonocarboxylic acid, fumaric acid, succinic acid, adipic acid,pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid,citric acid, glucaric acid, galactaric acid, amino acids, such asglutamic acid, aspartic acid, N-methylglycine, acetylaminoacetic acid,N-acetylasparagine or N-acetyl-cysteine, pyruvic acid, acetoacetic acid,phosphoserine, 2- or 3-glycerophosphoric acid, glucose-6-phosphoricacid, glucose-1-phosphoric acid, fructose-1,6-bisphosphoric acid, maleicacid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid,adamantanecarboxylic acid, benzoic acid, salicylic acid, 1- or3-hydroxynaphthyl-2-carboxylic acid, 3,4,5-trimethoxybenzoic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, 4-amino-salicylic acid,phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid,glucuronic acid, galacturonic acid, methanesulfonic or ethanesulfonicacid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,1,5-naphthalenedisulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid,methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid,N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamicacid, or other organic protonic acids, such as ascorbic acid.

When negatively charged radicals are present, such as tetrazolyl in R₄,salts may also be formed with bases, e.g. metal or ammonium salts, suchas alkali metal or alkaline earth metal salts, e.g. sodium, potassium,magnesium or calcium salts or ammonium salts with ammonia or suitableorganic amines, such as tertiary monoamires, e.g. triethylamine ortri-(2-hydroxyethyl)-amine, or heterocyclic bases e.g.N-ethyl-piperidine or N,N′-dimethyl-piperazine.

For the purposes of isolation or purification it is also possible to usepharmaceutically unsuitable salts, for example picrates or perchlorates.Only the pharmaceutically acceptable salts or the free compounds(optionally in the form of pharmaceutically compositions) are usedtherapeutically and they are therefore preferred.

In view of the close relationship between the novel compounds in freeform and in the form of their salts, including those salts that can beused as intermediates, for example in the purification of the novelcompounds or for identifying them, hereinbefore and hereinafter anyreference to the free compounds should be understood as including thecorresponding salts as appropriate and expedient.

The compounds of formula I have valuable pharmacological properties.They have anti-retroviral activity, especially against the viruses HIV-1and HIV-2 which are regarded as causes of AIDS, and may surprisinglyexhibit synergistic effects in combination with other compounds that areactive against retroviral aspartate proteases. The compounds of formulaI are inhibitors of retroviral aspartate proteases, especiallyinhibitors of the aspartate protease of HIV-1 or also HIV-2 and aretherefore suitable for the treatment of retroviral diseases, such asAIDS or its in preliminary stages (e.g. ARDS). Compounds of formula Ialso exhibit activity against corresponding animal retroviruses, such asSIV (in apes) or FIV (in cats).

Compounds of formula I exhibit, surprisingly, especially advantageousand important pharmacological properties, for example a very highantiviral activity in cell tests against various virus strains,including those which are resistant to other protease inhibitors, forexample in MT2-cells, good pharmacokinetics, such as highbioavailability, high selectivity and, especially, high blood levels(even in the case of oral administration).

The inhibitory action of the compounds of formula I on the proteolyticactivity of HIV-1-protease can be shown, for example, according to knownprocedures (see A. D. Richards et al., J. Biol. Chem. 265(14), 7733-7736(1990)). In that method the inhibition of the action of HIV-1-protease(preparation: see S. Billich et al., J. Biol. Chem. 263(34), 17905-17908(1990)) is measured in the presence of the icosapeptideRRSNQVSQNYPIVQNIQGRR (a synthetic substrate of HIV-1-protease, preparedby peptide synthesis in accordance with known procedures (see J.Schneider et al., Cell 54, 363-368 (1988)), which contains as substrateanalogue one of the cleavage sites of the gag-precursor protein (naturalsubstrate of HIV-1-protease). That substrate and its cleavage productsare analysed by high performance liquid chromatography (HPLC).

The test compound is dissolved in dimethyl sulfoxide. The enzymatic testis carried out by adding suitable dilutions of the inhibitor in 20 mMβ-morpholinoethanesuifonic acid (MES) buffer pH 6.0 to the test mixture.That mixture consists of the above-mentioned icosapeptide (122 μM) in 20mM MES-buffer pH 6.0. 100 μl are used per test batch. The reaction isstarted by the addition of 10 ml of HIV-1-protease solution and isstopped after one hour's incubation at 37° C. by the addition of 10 μlof 0.3M HClO₄. After centrifugation of the sample at 10 000×g for 5minutes, 20 μl of the resulting supernatant are applied to a 125×4.6 mmNucleosil® C18-5m-HPLC column (reversed-phase material supplied byMacherey & Nagel, Düren, FRG, based on silica gel that has been chargedwith C₁₈alkyl chains). The uncleaved icosapeptide and its cleavageproducts are eluted from the column by means of the following gradient:100% eluant 1→50% eluant 1+50% eluent 2 (eluant 1:10% acetonitrile, 90%H₂O, 0.1% trifluoroacetic acid (TFA); eluant 2:75% acetonitrile, 25%H₂O, 0.08% TFA) for 15 minutes, throughflow rate 1 ml/min. Thequantification of the eluted peptide fragments is carried out bymeasuring the peak height of the cleavage product at 215 nm.

Compounds of formula I exhibit inhibitory actions in the nanomolarrange; they preferably exhibit IC₅₀ values (IC₅₀=that concentrationwhich brings about a 50% reduction in the activity of HIV-1-protease incomparison with a control without inhibitor) of approximately 2×10⁻⁷ to5×10⁻⁹ M, preferably 5×10⁻⁸ to 5×10⁻⁹ M.

An alternative method (see Matayoshi et al., Science 247, 954-958(1990), here modified) of determining the inhibitory action againstHIV-1-protease may be described briefly as follows: the protease(purification: see Leuthardt et al., FEBS Lett. 326, 275-80 (1993)) isincubated at room temperature in 100 μl of assay buffer (20 mM MES pH6.0; 200 mM NaCl; 1 mM dithiothreitol; 0.01% polyethylene glycol(average molecular weight 6000 to 8000 da) with 10 μM fluorogenicsubstrate SC4400(4-(4-dimethylaminophenylazo)benzoyl-γ-aminobutyryl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS(EDANS=5-(2-aminoethylamino)-1-naphthalenesulfonic acid); NeosystemLaboratoire, France). The reaction is discontinued by the addition of900 μl of 0.03M HClO₄. The HIV-1-protease activity is determined bymeasuring the increase in fluorescence at λex=336, λem=485 nm. The IC₅₀values of compounds of formula I are determined as the concentration ofthe compound that is necessary to inhibit the protease activity in theassay by 50%. The numerical values are obtained from computer-generatedgraphs from data relating to at least 5 concentrations of the compoundof formula I in question with threefold determination per concentration.

In a further test it can be shown that compounds of formula I protectcells normally infected by HIV from such an infection or at least slowdown such an infection. For this test, MT-2-cells infected with HIV-1/MNare used. MT-2-cells have been transformed with HTLV-1 (a virus causingleukemia) and a continuous producer thereof; they are thereforeespecially sensitive to the cytopathogenic effect of HIV. MT-2-cells canbe obtained via the AIDS Research and Reference Reagent Program,Division of AIDS, NIAID, NIH from Dr. Douglas Richman (see J. Biol.Chem. 263, 5870-5875 (1988) and also Science 229, 563-566 1985)). TheMT-2-cells are cultured in RPMI 1640-medium (Gibco, Scotland; RPMIcomprises an amino acid mixture without glutamine) supplemented with 10%heat-inactivated foetal calf serum, glutamine and standard antibiotics.In all cases the cells, and also the virus stock solution used for theinfection (HIV-1/MN), are free of mycoplasms. The virus stock solutionis prepared as a cell culture supernatant of the permanently infectedcell line H9/HIV-1/MN, which can likewise be obtained via the AIDSResearch and Reference Program, Division of AIDS, NIAID, NIH from Dr.Robert Gallo (see also Science 224, 500-503 (1984) and Science 226,1165-1170 (1984)). The titre of the HIV-1/MN virus stock solution(determined by titration onto MT-2-cells) is 4.2×10⁵ TCID50/ml(TCID50=Tissue Culture Infective Dose=dose that infects 50% of theMT-2-cells). In order to measure the infection-inhibiting action of thecompounds of formula I, 50 μl of the test compound in question inculture medium and 2800 TCID50 of HIV-1/MN in 100 μl of culture mediumare added to 2×10⁴ exponentially growing MT-2-cells which have beenapplied in 50 μl of culture medium to 96-well microtitre plates (havinga round base). After 4 days' incubation (at 37° C., 5% CO₂) a 10 μlsample of the supernatant is taken from each well, transferred to afurther 96-well microtitre plate and (if necessary) stored at −20° C. Inorder to measure the activity of the virus-associated reversetranscriptase, 30 μl of reverse transcriptase (RT) cocktail are added toeach sample. The reverse transcriptase cocktail consists of 50 mM Tris(α,α,α-tris(hydroxymethyl)methylamine, Ultra pur, Merck, Germany) pH7.8; 75 mM KCl, 2 mM dithiothreitol, 5 mM MgCl₂; 0.1% Nonidet P-40(detergent; Sigma, Switzerland), 0.8 mM EDTA, 10 μg/ml Poly-A(Pharmacia, Uppsala, Sweden) and 0.16 μg/ml oligo(T) (=pdT(12-18),Pharmacia, Uppsala, Sweden) as “template primer”—if desired, the mixtureis filtered through a 0.45 mm Acrodisc filter (Gelman Sciences Inc., AnnArbor, USA). It is stored at −20° C. Prior to the test, 0.1% (v/v)[alpha-³²P]dTTP is added to aliquots of the solution in order toestablish a radioactivity of 10 μCi/ml.

After mixing, the plate is incubated for 2 hours at 37° C. 5 μl of thereaction mixture are transferred to DE81 paper (Whatman, one filter perwell). The dried filters are washed three times for 5 minutes with 300mM NaCl/25 mM trisodium citrate and then once with ethanol and againdried in the air. The radioactivity on the filters is measured in aMatrix Packard 96-well counter (Packard, Zurich, Switzerland). The ED₉₀values are calculated and are defined as the concentration of the testcompound that reduces the RT activity by 90% in comparison with acontrol without test compound.

The preferred compounds of formula I here exhibit an ED₉₀, that is tosay a 90% inhibition of virus replication, at concentrations of from10⁻⁷ to 10⁻⁹M, especially from 5×10⁻⁹ to 10⁻⁹M.

Accordingly, the compounds of formula I are suitable for the highlyeffective retardation of the replication of HIV-1 in cell cultures.

In order to determine their pharmacokinetics, the compounds of formula Iare dissolved in dimethyl sulfoxide (DMSO) in a concentration of 240mg/ml. The resulting solutions are diluted 1:20 (v/v) with 20% (w/v)aqueous hydroxypropyl-β-cyclodextrin solution in order to obtain aconcentration of the test compound in question of 12 mg/ml. Theresulting solution is treated briefly with ultrasound and administeredorally to female BALB/c mice (Bomholt-garden, Copenhagen, Denmark) byartificial tube feeding at a dose of 120 mg/kg. At fixed times (forexample 30, 60, 90, 120 min) after administration, mice are sacrificedand the plasma stored in heparinised test tubes. The blood iscentrifuged (12 000×g, 5 min) and the plasma removed. The plasma isdeproteinised by the addition of an equal volume of acetonitrile. Themixture is mixed using a vortex mixer and and left to stand at roomtemperature for 20 to 30 minutes. The precipitate is pelleted bycentrifugation (12 000×g, 5 min), and the concentration of the testcompound is determined by reversed phase high performance liquidchromatography (HPLC).

The HPLC analysis of the samples obtained in accordance with the methoddescribed above is carried out on a 125×4.6 mm Nucleosil® C₁₈-column(reversed-phase material supplied by Macherey & Nagel, Düren, Germany,based on silica gel derivatised with carbon radicals having 18 carbonatoms), using a 2 cm long preliminary column of the same columnmaterial. The test is carried out with the following linearacetonitrile/water gradient (in each case in the presence of 0.05%trifluoroacetic acid): 20% acetonitrile to 100% acetonitriie for 20 min;then 5 min 100% acetonitrile; then returning to the initial conditionsfor 1 min and 4 min reequilibration. The flow rate is 1 ml/min. Underthose conditions the compound of formula I from Example 1, for example,has a retention time of about 15.5 minutes, and its detection limit is0.1-0.2 μM. The test compound is detected by UV absorption measurementat 255 nm. Peaks are identified by the retention time and the UVspectrum between 205 and 400 nm. The concentrations are determined bythe external standard method; the peak heights are obtained fordetermining the concentrations by comparison with standard curves. Thestandard curves are obtained by analogous HPLC analysis of mouse plasmathat contains known concentrations of the test compound in question andthat has been worked up in accordance with the method described above.

In that experiment compounds of formula I produce plasma concentrationsfar above the ED₉₀ determined above in the cell experiment, for exampleup to 8000 times greater than the ED₉₀ after 30 minutes and up to 10 500times greater than the ED₉₀ after 90 minutes, preferably plasmaconcentrations of from 0.1 μM to 25 μM, especially from 1 to 25 μM, 30minutes after oral administration, and plasma concentrations of from 0.5to 35 μM, especially from 1 to 35 μM, 90 minutes after oraladministration.

Analogously, in dogs, the blood level of the compounds of formula I, forexample the title compound of Example 46, can be measured, for example,using the formulations according to either Example 63 or Example 64,there being used, for example, from 92 to 100 mg/kg of the compoundwhich is administered by stomach tube, the blood levels then beingmeasured, e.g. 1, 2, 3, 4, 6, 8 and 24 hours after administration. Here,also, blood levels in the micromolar range can be found.

In particular, the combination of high bioavailability (high plasmalevels), which is surprising in itself, and unexpectedly excellent ED₉₀in the cell experiment renders the compounds of the present inventionvaluable in an unforeseen way. Activity against inhibitors of retroviralaspartate proteases to which resistance has already developed is alsostill possible and is a further important advantage of the compoundsaccording to the invention.

That can be demonstrated, for example, by the following or analogoustests:

Inhibitor-resistant HIV-1 protease variants are cloned as follows:

By way of PCR-supported mutagenesis and cloning, HIV-1 protease mutantsare generated that are based on the infectious clone pNL4-3 (freelyavailable via the “NIH AIDS reference and reagent program”, the originalreference is A. Adachi et al. J. Virol (1986) 59, 284-91—but it can, ofcourse, be any other HIV clone, or even clinical material, provided thatcomparability is ensured). Those otherwise isogenic point mutantscontain only those changes which have been described in publications inconnection with viral resistance to various protease inhibitors. Thecloned fragments are, for example, only 500 base pairs in length, all ofthe remainder being unchanged. By using mutations in always the sameclone, direct comparability is ensured, which would rot be the case in adirect comparison of clinical samples or of different HIV clones. In thetransient DNA transfection assay in human T4-positive cells (HeLaT4),the resulting proviruses also demonstrate the finding of reducedinhibitor activity in comparison with the wild type virus, that is tosay increased resistance.

That system is used as a transient DNA transfection system for tests:

1) in order to identify possible cross-resistances of protease variantsto several protease inhibitors; and

2) in order to establish the potency and resistance profile of novelinhibitor candidates.

For example, in the said transfection system1-[4-(pyridin-2-yl)phenyl]-4(S)-hydroxy-5(S)-2,5-bis-[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane(Example 46) has an in vitro potency that, with an IC90 of <30 nM, is inpractical terms better than that of saquinavir (Hoffmann-LaRoche, seebelow) and the activity against a resistant variant (45I,76F strain)which has been established against5(S)-(tert-butoxycarbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(2,3,4-trimethoxyphenylmethyl)-hexanoyl-(L)-valyl-N-(2-methoxy-ethyl)-amide(=Lasinavir, see EP 0 708 085, published on Apr. 24, 1996; Novartis AG,originally Ciba-Geigy AG), is comparable with saquinavir and better thanthat of indinavir (Merck & Co., Inc., see below) or ritonavir (Abbott,see below). Compared with other strains (e.g. 46I/47V/50V (VX478)), 1 nMproduced an activity that was more potent (not quantified) than that ofsaquinavir, ritonavir and indinavir. Instead of the strains mentioned,there may be used any human T4-positive cells, such as the HeLa T4cells, deposited under that name by Richard Axel and Paul Maddon in “NIHAIDS reference and reagent program” and obtainable via that source.

In principle, the relevant mutations for the above test systems forresistances are known (see e.g. relating to the 48V/90M strain(saquinavir resistance): Jacobsen, H., Yasargil, K., Winslow, D. L.,Craig, J. C., Krohn, A., Duncan, I. B., & Mous, J. Virology 206, 527(1995); Merck Mutationen (several, e.g. 71V/82T/84V): Condra, J. H.,Schleif, W. A., Blahy, O. M., Gabryelski, L. J., Graham, D. J.,Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M.,& et al Nature 374, 569 (1995); Abbott 82V/84A strain: Markowitz, M.,Mo, H., Kempf, D. J., Norbeck, D. W., Bhat, T. N., Erickson, J. W., &Ho, D. D. J. Virol. 69, 701 (1995).

In the determination of the anti-enzymatic activity against numeroushuman aspartate proteases in accordance with known methods (see, forexample, Biochem. J. 265, 871-878 (1990)), compounds of formula Iexhibit a high selectivity towards the retroviral aspartate protease ofHIV, especially HIV-1. For example, the inhibition constant (IC₅₀) forcompounds of formula I in the test against cathepsin D is more than 10μM, especially more than 25 μM. The IC₅₀ against human cathepsin D inthat test is measured at pH 3.1. The test is carried out in accordancewith known procedures using the substrate KPIQF*NphRL (see Jupp, R. A.,Dunn, B. M., Jacobs, J. W., Viasuk, G., Arcuri, K. E., Veber, D. F., S.Perow, D. S., Payne, L. S., Boger, J., DeLazio, S., Chakrabarty, P. K.,TenBroeke, J., Hangauer, D. G., Ondeyka, D., Greenlee, W. J. and Kay,J.: The selectivity of statine-based inhibitors against various humanaspartic proteases. Biochem. J. 265: 871-878 (1990)).

The compounds of formula I can be used alone or in combination (as a setcombination of corresponding compositions or as a combination ofindividual compounds or individual compositions in a time-staggeredsequence) with one or more other pharmaceutically active substances (orsalts thereof provided that at least one salt-forming group is present)that are effective against retroviruses, especially HIV, such as HIV-1or HIV-2; especially with inhibitors of reverse transcriptase, moreespecially nucleoside analogues, especially 3′-azido-3′-deoxypyrimidine(=zidovudine=®RETROVIR, Burroughs-Wellcome), 2′,3′-dideoxycytidine(=zalcitabine=®HIVID, Hoffmann-LaRoche), 2′,3′-dideoxyinosine(=didanosine=®VIDEX, Bristol-Myers-Squibb) or(2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one(=lamivudine, Glaxo); especiallyd4C=2′,3′-didehydro-2′,3′-dideoxycytidine,d4T=2′,3′-didehydro-2′,3′-dideoxythymidine (=stavudine=®ZERIT) or2′,3′-dideoxyinosine (=ddlno=DZI=didanosine=®VIDEX); or non-nucleosideanalogues, such as11-cyclopropyl-5,11-dihydro-4-methyl-(6H)-dipyrido[3,2-b;2′,3′-e]-[1,4]-diazepin-6-one;or with one or more (especially one or also two) other inhibitors ofretroviral aspartate proteases, especially aspartate proteases of HIV,such as HIV-1 and HIV-2, especially

a) one of the inhibitors mentioned in EP 0 346 847 (published on Dec.20, 1989) and EP 0 432 695 (published on Jun. 19, 1991; corresponds toU.S. Pat. No. 5,196,438, published on Mar. 23, 1993), especially thecompound designated Ro 31-8959 (=saquinavir; Hoffmann-LaRoche);

b) one of the inhibitors mentioned in EP 0 541 168 (published on Dec.12, 1993; corresponds to U.S. Pat. No. 5,413,999), especially thecompound designated L-735,524 (=indinavir=®CRIXIVAN; Merck & Co., Inc.);

c) one of the inhibitors mentioned in EP 0 486 948 (published on May 27,1992; corresponds to U.S. Pat. No. 5,354,866), especially the compounddesignated ABT-538 (=ritonavir; Abbott);

d) the compound designated KVX-478 (or VX-478 or 141W94; GlaxoWellcome,Vertex and Kissei Pharmaceuticals)

e) the compound designated AG-1343 (Agouron);

f) the compound designated KNI-272 (Nippon Mining);

g) the compound designated U-96988 (Upjohn);

h) the compound designated BILA-2011 BS (=palinavir;Boehringer-Ingelheim), and/or

l) the compound5(S)-(tert-butoxycarbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-(2,3,4-trimethoxyphenylmethyl)-hexanoyl-(L)-valyl-N-(2-methoxy-ethyl)-amide(=lasinavir, see EP 0 708 085, published on Dec. 24, 1996; Novartis AG,originally Ciba-Geigy AG),

or in each case a salt thereof, provided that salt-forming groups arepresent.

The compounds of formula I can also be used in the prevention, controland treatment of retrovirus infections, especially HIV, such as HIV-1 orHIV-2, in cell cultures, especially cell cultures of lymphocyte celllines, from warm-blooded animals, which is advantageous especially inthe case of very valuable cell cultures that produce, for example,specific antibodies, vaccines or messenger substances, such asinterleukins and the like, and are therefore of great commercial value.

Finally, the compounds of formula I can be used as standards inexperiments, for example as HPLC standards or as standards for thecomparison of animal models in respect of different aspartate proteaseinhibitors, for example in respect of the blood levels achievable.

In the groups of preferred compounds of formula I mentioned below, it ispossible where expedient (for example in order to replace more generaldefinitions by more specific definitions or, especially, by definitionsdescribed as being preferred) to use definitions of substituents fromthe general definitions given above; in each case preference is given tothe definitions described above as being preferred or given as examples.

Preference is given to a compound of formula I, especially of formulaIa, wherein

R₁ is lower alkoxycarbonyl, especially methoxycarbonyl orethoxycarbonyl,

R₂ is isopropyl, sec-butyl (preferably in the (S)-configuration), ortert-butyl,

R₃ is phenyl or also cyclohexyl,

R₄ is phenyl substituted in the 4-position by one of the followingradicals bonded by way of a ring carbon atom: thienyl (=thiophenyl);oxazolyl; thiazolyl; imidazoyl; 1,4-thiazinyl; triazolyl that isunsubstituted or, especially, substituted by 1-methyl-1-phenyl-ethyl orpreferably by tert-butyl or especially by methyl, such as 1-, 2- or 4-(methyl or tert-butyl)-triazol-3-yl; tetrazolyl that is unsubstitutedor, especially, substituted by 1-methyl-1-phenyl-ethyl or preferably bylower alkyl, such as by tert-butyl or especially by methyl, such as2H-tetrazol-5-yl substituted by 1-methyl-1-phenyl-ethyl or preferably bylower alkyl, such as by tert-butyl or especially by methyl, or1H-tetrazol-5-yl substituted by methyl; pyridinyl; prazinyl; andpyrimidinyl; especially 2- or 3-thienyl (=thiophen-2-yl orthiophen-3-yl); thiazol-5-yl; thiazol-2-yl; 2H-tetrazol-5-yl that isunsubstituted or, especially, substituted in the 2-position by1-methyl-1-phenyl-ethyl or preferably by tert-butyl or especially bymethyl; 1H-tetrazol-5-yl substituted in the 1-position by methyl;pyridin-2-yl; pyridin-3-yl; pyridin-4-yl; or pyrazin-2-yl;

R₅ is isopropyl, sec-butyl (preferably in the (S)-configuration),tert-butyl or methylthiomethyl, and

R₆ is lower alkoxycarbonyl, especially methoxycarbonyl orethoxycarbonyl,

or a salt thereof (especially a pharmaceutically acceptable saltthereof), provided that at least one salt-forming group is present.

Greater preference is given to a compound of formula I, wherein

R₁ is methoxycarbonyl or ethoxycarbonyl,

R₂ is isopropyl, sec-butyl or tert-butyl,

R₃ is phenyl,

R₄ is phenyl substituted in the 4-position of the phenyl ring by 2- or3-thienyl (=thiophen-2-yl or thiophen-3-yl); thiazol-5-yl; thiazol-2-yl;2H-tetrazol-5-yl that is unsubstituted or, especially, substituted inthe 2-position by 1-methyl-1-phenyl-ethyl or preferably by tert-butyl orespecially by methyl; 1H-ietrazol-5-yl substituted in the 1-position bymethyl; pyridin-2-yl; pyridin-3-yl; pyridin-4-yl; or by pyrazin-2-yl;and is especially 4-(thiazol-2-yl)-phenyl; 4-(thiazol-5-yl)-phenyl;4-(pyridin-2-yl)-phenyl; or 4-(2-methyl-tetrazol-5-yl)-phenyl;

R₅ is isopropyl, sec-butyl, tert-butyl or methylthiomethyl; and

R₆ is methoxycarbonyl or ethoxycarbonyl;

with the proviso that at least one of the two radicals R₂ and R₅ istert-butyl, provided that R₄ is phenyl substituted in the 4-position ofthe phenyl ring by 2- or 3-thienyl (=thiophen-2-yl or thiophen-3-yl);thiazol-5-yl; thiazol-2-yl; 2H-tetrazol-5-yl that is unsubstituted or,especially, substituted in the 2-position by 1-methyl-1-phenyl-ethyl orpreferably by tert-butyl or especially by methyl; 1H-tetrazol-5-ylsubstituted in the 1-position by methyl; pyridin-3-yl; pyridin-4-yl; orby pyrazin-2-yl;

or a (preferably pharmaceutically acceptable) salt thereof, providedthat at least one salt-forming group is present.

Special preference is given to a compound of formula I, wherein

R₁ is methoxycarbonyl or ethoxycarbonyl,

R₂ is isopropyl, sec-butyl or tert-butyl,

R₃ is phenyl,

R₄ is 4-(thiazol-2-yl)-phenyl, 4-(thiazol-5-yl)-phenyl,4-(pyridin-2-yl)-phenyl or 4-(2-methyl-tetrazol-5-yl)-phenyl;

R₅ is isopropyl, sec-butyl, tert-butyl or methylthiomethyl; and

R₆ is methoxycarbonyl or ethoxycarbonyl;

or a (preferably pharmaceutically acceptable) salt thereof, providedthat at least one salt-forming group is present.

Each of the compounds of formula I mentioned below, or a (preferablypharmaceutically acceptable) salt thereof, is highly preferred:

1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane;

1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis-[N-(N-methoxycarbonyl-(L)-tert-leucyl)-amino]-6-phenyl-2-azahexane;

1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane;

1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-S-methylcysteinyl)-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane;

1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-ethoxycarbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane;

1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane;

1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane;

1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis-[N-(N-methoxycarbonyl-(L)-tert-leucyl)-amino]-6-phenyl-2-azahexane;

1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane;

1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane;

1-[4-(2-methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis-[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane;

1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis-[N-(N-methoxycarbonyl-(L)-valyl)amino]-6-phenyl-2-azahexane;

1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis-[N-(N-methoxycarbonyl-(L)-tert-leucyl)-amino]-6-phenyl-2-azahexane;

1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane;or

1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane.

Special preference is given to the compounds of formula I mentioned inthe Examples, or to pharmaceutically acceptable salts thereof providedthat at least one salt-forming group is present.

The compounds of formula I and salts of those compounds having at leastone salt-forming group are prepared according to processes known per se,for example as follows:

a) a hydrazine derivative of formula

wherein the radicals R₄, R₅ and R₆ are as defined for compounds offormula I, is added to an epoxide of formula IV*,

especially of formula IV,

wherein the radicals R₁, R₂ and R₃ are as defined for compounds offormula I, free functional groups with the exception of thoseparticipating in the reaction being, if necessary, in protected form,and any protecting groups are removed, or

b) an amino compound of formula V*,

especially of formula V

wherein the radicals R₁, R₂, R₃ and R₄ are as defined for compounds offormula I, is condensed with an acid of formula

or with a reactive acid derivative thereof, wherein the radicals R₅ andR₆ are as defined for compounds of formula I, free functional groupswith the exception of those participating in the reaction being, ifnecessary, in protected form, and any protecting groups are removed, or

c) an amino compound of formula VII*,

especially of formula VII

wherein the radicals R₃, R₄, R₅ and R₆ are as defined for compounds offormula I, is condensed with an acid of formula

or with a reactive acid derivative thereof, wherein R₁ and R₂ are asdefined for compounds of formula I, free functional groups with theexception of those participating in the reaction being, if necessary, inprotected form, and any protecting groups are removed, or

d) to prepare a compound of formula I wherein the pairs of substituentsR₁ and R₆ and R₂ and R₅ are in each case two identical radicals, asdefined for compounds of formula I, and R₃ and R₄ are as defined forcompounds of formula I, a diamino compound of formula IX*

especially of formula IX,

wherein the radicals are as just defined, is condensed with an acid offormula

or with a reactive acid derivative thereof, wherein R₁′ and R₂′ are asdefined for R₁ and R₆ and for R₂ and R₅ respectively, in formula I, thepairs R₁ and R₆ and R₂ and R₅ being in each case two identical radicals,free functional groups with the exception of those participating in thereaction being, if necessary, in protected form, and any protectinggroups are removed, or

e) an imino compound of formula (I′)*,

especially of formula I′

wherein the radicals R₁, R₂, R₃, R₅ and R₆ are as defined for compoundsof formula I, is reacted with a compound of formula X,

wherein X is a leaving group and R₄ is as defined for compounds offormula I, free functional groups with the exception of thoseparticipating in the reaction being, if necessary, in protected form,and any protecting groups are removed, or

f) an imino compound of formula (I′)*,

especially of formula I′

wherein the radicals R₁, R₂, R₃, R₅ and R₆ are as defined for compoundsof formula I, is reacted with an aldehyde of formula X*,

wherein R₄ is as defined for compounds of formula I, or with a reactivederivative thereof, with reductive alkylation, free functional groupswith the exception of those participating in the reaction being, ifnecessary, in protected form, and any protecting groups are removed,

and, if desired, a compound of formula I having at least onesalt-forming group obtainable in accordance with any one of processes a)to f) above is converted into its salt or an obtainable salt isconverted into the free compound or into a different salt and/orisomeric mixtures which may be obtainable are separated and/or acompound of formula I according to the invention is converted into adifferent compound of formula I according to the invention.

The above processes are described in more detail below with reference topreferred embodiments.

In the following description of the individual processes and thepreparation of the starting materials, unless otherwise indicated theradicals R₁, R₂, R₃, R₄, R₅ and R₆ are as defined for compounds offormula I, preference being given in each case to the definitions givenas being preferred.

Process a) (Addition of an amine to an epoxide):

In the hydrazine derivatives of formula III, the amino groupparticipating in the reaction preferably has a free hydrogen atom; itmay, however, itself have been derivatised in order to increase thereactivity of the hydrazine derivative.

The epoxide of formula IV enables the terminal addition of the hydrazinederivative to proceed preferentially.

In starting materials, functional groups the reaction of which is to beavoided, especially carboxy, amino and hydroxy groups, can be protectedby suitable protecting groups (conventional protecting groups) which arecustomarily used in the synthesis of peptide compounds, and also in thesynthesis of cephalosporins and penicillins as well as nucleic acidderivatives and sugars. Those protecting groups may already be presentin the precursors and are intended to protect the functional groups inquestion against undesired secondary reactions, such as acylation,etherification, esterification, oxidation, solvolysis and the like. Incertain cases the protecting groups can additionally cause reactions toproceed selectively, for example stereoselectively. It is characteristicof protecting groups that they can be removed easily, i.e. withoutundesired secondary reactions taking place, for example by solvolysis,reduction, photolysis, and also enzymatically, for example also underphysiological conditions. Radicals analogous to protecting groups mayalso be present in the end products, however. Compounds of formula Ihaving protected functional groups may have greater metabolic stabilityor pharmacodynamic properties that are better in some other way than thecorresponding compounds having free functional groups. Hereinabove andhereinbelow, protecting groups are referred to in their true sense whenthe radicals in question are not present in the end products.

The protection of functional groups by such protecting groups, theprotecting groups themselves and the reactions for their removal aredescribed, for example, in standard works such as J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, London and NewYork 1973, in Th. W. Greene, “Protective Groups in Organic Synthesis”,Wiley, New York 1981, in “The Peptides”, Volume 3 (E. Gross and J.Meienhofer, eds.), Academic Press, London and New York 1981, in“Methoden der organischen Chemie” (“Methods of Organic Chemistry”),Houben-Weyl, 4th edition, Volume 15/l, Georg Thieme Verlag, Stuttgart1974, in H.-D. Jakubke and H. Jescheit, “Aminosäuren, Peptide, Proteine”(“Amino acids, peptides, proteins”), Verlag Chemie, Weinheim, DeerfieldBeach and Basle 1982, and in Jochen Lehmann, “Chemie der Kohlenhydrate:Monosaccharide und Derivate” (“The Chemistry of Carbohydrates:monosaccharides and derivatives”), Georg Thieme Verlag, Stuttgart 1974.

A carboxy group is protected, for example, in the form of an ester groupwhich can be cleaved selectively under mild conditions. A carboxy groupprotected in esterified form is esterified especially by a lower alkylgroup that is preferably branched in the 1-position of the lower alkylgroup or substituted in the 1- or 2-position of the lower alkyl group bysuitable substituents.

A protected carboxy group esterified by a lower alkyl group is, forexample, methoxycarbonyl or ethoxycarbonyl.

A protected carboxy group esterified by a lower alkyl group that isbranched in the 1-position of the lower alkyl group is, for example,tert-lower alkoxycarbonyl, for example tert-butoxycarbonyl.

A protected carboxy group esterified by a lower alkyl group that issubstituted in the 1- or 2-position of the lower alkyl group by suitablesubstituents is, for example, arylmethoxycarbonyl having one or two arylradicals, wherein aryl is phenyl that is unsubstituted or mono-, di- ortri-substituted, for example, by lower alkyl, for example tert-loweralkyl, such as tert-butyl, lower alkoxy, for example methoxy, hydroxy,halogen, for example chlorine, and/or by nitro, for examplebenzyloxycarbonyl, benzyloxycarbonyl substituted by the mentionedsubstituents, for example 4-nitrobenzyloxycarbonyl or4-methoxybenzyloxy-carbonyl, diphenylmethoxycarbonyl ordiphenylmethoxycarbonyl substituted by the mentioned substituents, forexample di(4-methoxyphenyl)methoxycarbonyl, and also carboxy esterifiedby a lower alkyl group, the lower alkyl group being substituted in the1- or 2-position by suitable substituents, such as 1-lower alkoxy-loweralkoxycarbonyl, for example methoxymethoxycarbonyl,1-methoxyethoxycarbonyl or 1-ethoxyethoxycarbonyl, 1-loweralkylthio-lower alkoxycarbonyl, for example 1-methylthiomethoxycarbonylor 1-ethylthioethoxycarbonyl, aroylmethoxycarbonyl wherein the aroylgroup is benzoyl that is unsubstituted or substituted, for example, byhalogen, such as bromine, for example phenacyloxycarbonyl, 2-halo-loweralkoxycarbonyl, for example 2,2,2-trichloroethoxycarbonyl,2-bromoethoxycarbonyl or 2-iodoethoxycarbonyl, as well as2-(tri-substituted silyl)-lower alkoxycarbonyl wherein the substituentsare each independently of the others an aliphatic, araliphatic,cycloaliptatic or aromatic hydrocarbon raodical that is unsubstituted orsubstituted, for example, by lower alkyl, lower alkoxy, aryl, halogenand/or by nitro, for example lower alkyl, phenyl-lower alkyl, cycloalkylor phenyl each of which is unsubstituted or substituted as above, forexample 2-tri-lower alkylsilyl-lower alkoxycarbonyl, such as 2-tri-loweralkylsilylethoxycarbonyl, for example 2-trimethylsilylethoxycarbonyl or2-(di-n-butyl-methyl-silyl)-ethoxycarbonyl, or2-triarylsilylethoxycarbonyl, such as triphenylsilylethoxycarbonyl.

A carboxy group may also be protected in the form of an organicsilyloxycarbonyl group. An organic silyloxycarbonyl group is, forexample, a tri-lower alkylsilyloxycarbonyl group, for exampletrimethylsilyloxycarbonyl.

A protected carboxy group is preferably tert-lower alkoxycarbonyl, forexample tert-butoxy-carbonyl, benzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl ordiphenylmethoxycarbonyl.

A protected amino group may be protected by an amino-protecting group,for example in the form of an acylamino, arylmethylamino, etherifiedmercaptoamino, 2-acyl-lower alk-1-enylamino or silylamino group, or inthe form of an azido group.

In a corresponding acylamino group, acyl is, for example, the acylradical of an organic carboxylic acid having, for example, up to 18carbon atoms, especially an unsubstituted or substituted, for examplehalo- or aryl-substituted, lower alkanecarboxylic acid or anunsubstituted or substituted, for example halo-, lower alkoxy- ornitro-substituted, benzoic acid, or, preferably, of a carbonic acidsemiester. Such acyl groups are, for example, lower alkanoyl, such asformyl, acetyl, propionyl or pivaloyl, halo-lower alkanoyl, for example2-haloacetyl, such as 2-chloro-, 2-bromo-, 2-iodo-, 2,2,2-trifluoro- or2,2,2-trichloro-acetyl, unsubstituted or substituted, for example halo-,lower alkoxy- or nitro-substituted, benzoyl, such as benzoyl,4-chlorobenzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl, loweralkoxycarbonyl, preferably lower alkoxycarbonyl that is branched in the1-position of the lower alkyl radical or suitably substituted in the 1-or 2-position, for example tert-lower alkoxycarbonyl, such astert-butoxycarbonyl, arylmethoxycarbonyl having one, two or three arylradicals which are phenyl that is unsubstituted or mono- orpoly-substituted, for example, by lower alkyl, especially tert-loweralkyl, such as tert-butyl, lower alkoxy, such as methoxy, hydroxy,halogen, such as chlorine, and/or by nitro, for examplebenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, diphenylmethoxycarbonyl,9-fluorenylmethoxycarbonyl or di(4-methoxyphenyl)methoxycarbonyl,aroylmethoxycarbonyl wherein the aroyl group is preferably benzoyl thatis unsubstituted or substituted, for example, by halogen, such asbromine, for example phenacyloxycarbonyl, 2-halo-lower alkoxycarbonyl,for example 2,2,2-trichloroethoxycarbonyl, 2-bromoethoxycarbonyl or2-iodoethoxycarbonyl, 2-(tri-substituted silyl)-lower alkoxycarbonyl,for example 2-tri-lower alkylsilyl-lower alkoxycarbonyl, such as2-trimethylsilylethoxycarbonyl or2-(di-n-butyl-methyl-silyl)-ethoxycarbonyl, or triarylsilyl-loweralkoxycarbonyl, for example 2-triphenylsilylethoxycarbonyl.

In an arylmethylamino group, for example a mono-, di- or especiallytri-arylmethylamino group, the aryl radicals are especiallyunsubstituted or substituted phenyl radicals. Such groups are, forexample, benzyl-, diphenylmethyl- or especially trityl-amino, or veryespecially 1-aryl-lower alkylmethylamino wherein the lower alkyl radicalis preferably branched in the 1-position, such as in1-methyl-1-phenyl-ethylamino. In an etherified mercaptoamino group themercapto group is especially in the form of substituted arylthio oraryl-lower alkylthio wherein aryl is, for example, phenyl that isunsubstituted or substituted, for example, by lower alkyl, such asmethyl or tert-butyl, lower alkoxy, such as methexy, halogen, such aschlorine, and/or by nitro, for example 4-nitrophenylthio.

In a 2-acyl-lower alk-1-enyl radical that can be used as anamino-protecting group, acyl is, for example, the corresponding radicalof a lower alkanecarboxylic acid, of a benzoic acid that isunsubstituted or substituted, for example, by lower alkyl, such asmethyl or tert-butyl, lower alkoxy, such as methoxy, halogen, such aschlorine, and/or by nitro, or especially of a carbonic acid semiester,such as a carbonic acid lower alkyl semiester. Corresponding protectinggroups are especially 1-lower alkanoyl-lower alk-1-en-2-yl, for example1-lower alkanoyl-prop-1-en-2-yl, such as 1-acetyl-prop-1-en-2-yl, orlower alkoxycarbonyl-lower alk-1-en-2-yl, for example loweralkoxycarbonyl-prop-1-en-2-yl, such as 1-ethoxycarbonyl-prop-1-en-2-yl.

A silylamino group is, for example, a tri-lower alkylsilylamino group,for example trimethylsilylamino or tert-butyl-dimethylsilylamino. Thesilicon atom of the silylamino group can also be substituted by only twolower alkyl groups, for example methyl groups, and by the amino group orcarboxy group of a second molecule of formula I. Compounds having suchprotecting groups can be prepared, for example, using the correspondingchlorosilanes, such as dimethylchlorosilane, as silylating agents.

An amino group can also be protected by conversion into the protonatedform; suitable corresponding anions are especially those of stronginorganic acids, such as sulfuric acid, phosphoric acid or hydrohalicacids, for example the chlorine or bromine anion, or of organic sulfonicacids, such as p-toluenesulfonic acid.

Preferred amino-protecting groups are lower alkoxycarbonyl, phenyl-loweralkoxycarbonyl, fluorenyl-lower alkoxycarbonyl, 2-lower alkanoyl-loweralk-1-en-2-yl, 1-methyl-1-phenyl-ethyl and lower alkoxycarbonyl-loweralk-1-en-2-yl.

A hydroxy group can be protected, for example, by an acyl group, forexample lower alkanoyl that is substituted by halogen, such as chlorine,such as 2,2-dichloroacetyl, or especially by an acyl radical of acarbonic acid semiester mentioned for protected amino groups. Apreferred hydroxy-protecting group is, for example,2,2,2-trichloroethoxycarbonyl, 4-nitrobenzyloxycarbonyl,diphenylmethoxycarbonyl or trityl. A hydroxy group can also be protectedby tri-lower alkylsilyl, for example trimethylsilyl, triisopropylsilylor tert-butyl-di-methylsilyl, a readily removable etherifying group, forexample an alkyl group, such as tert-lower alkyl, for exampletert-butyl, an oxa- or a thia-aliphatic or -cycloaliphatic, especially2-oxa- or 2-thia-aliphatic or -cycloaliphatic, hydrocarbon radical, forexample 1-lower alkoxy-lower alkyl or 1-lower alkylthio-lower alkyl,such as methoxymethyl, 1-methoxyethyl, 1-ethoxyethyl, methylthiomethyl,1-methylthioethyl or 1-ethylthioethyl, or 2-oxa- or 2-thia-cycloalkylhaving from 5 to 7 ring atoms, such as 2-tetrahydrofuryl or2-tetrahydropyranyl, or a corresponding thia analogue, and also by1-phenyl-lower alkyl, such as benzyl, diphenylmethyl or trityl, whereinthe phenyl radicals can be substituted, for example, by halogen, forexample chlorine, lower alkoxy, for example methoxy, and/or by nitro.

A hydroxy group and an amino group that are adjacent to one another in amolecule can be protected, for example, by bivalent protecting groups,such as a methylene group that is preferably substituted, for example byone or two lower alkyl radicals or by oxo, for example unsubstituted orsubstituted alkylidene, for example lower alkylidene, such asisopropylidene, cycloalkylidene, such as cyclohexylidene, a carbonylgroup or benzylidene.

In the context of this disclosure, a protecting group, for example acarboxy-protecting group, is to be understood as being expressly also apolymeric carrier that is bonded in a readily removable maiiner to thefunctional group, for example the carboxy group, to be protected, forexample a carrier suitable for the Merrifield synthesis. Such a suitablepolymeric carrier is, for example, a polystyrene resin weaklycross-linked by copolymerisation with divinylbenzene and carrying bridgemembers suitable for reversible bonding.

The addition of the compounds of formula III to the epoxides of formulaIV is carried out preferably under the reaction conditions customarilyused for the addition of nucleophiles to epoxides.

The addition is carried out especially in aqueous solution and/or in thepresence of polar solvents, such as alcohols, for example methanol,ethanol, isopropanol or ethylene glycol, ethers, such as dioxane,amides, such as dimethylformamide, or phenols, such as phenol, and alsounder anhydrous conditions, in non-polar solvents, such as benzene ortoluene, or in benzene/water emulsions, optionally in the presence ofacidic or basic catalysts, for example alkali hydroxide solutions, suchas sodium hydroxide solution, or in the presence of solid phasecatalysts doped with the hydrazine, such as aluminium oxide, in ethers,for example diethyl ether, generally at temperatures of fromapproximately 0° C. to the boiling temperature of the reaction mixturein question, preferably from 20° C. to reflux temperature, optionallyunder elevated pressure, for example in a bomb tube, in which case it isalso possible to exceed the boiling temperature measurable at normalpressure, and/or under an inert gas, such as nitrogen or argon, it beingpossible for each of the two compounds of formulae III and IV to bepresent in excess, for example in a molar ratio of from 1:1 to 1:100,especially in a molar ratio of from 1:1 to 1:10, more especially in aratio of from 1:1 to 1:3.

The freeing of protected groups may be effected in accordance with themethods described below under the heading “Removal of protectinggroups”.

Process b) (Formation of an amide bond)

In starting materials of formulae V and VI, functional groups, with theexception of groups that are to participate in the reaction or that donot react under the reaction conditions, are protected independently ofone another by one of the protecting groups mentioned under Process a).

The compounds of formula VI either contain a free carboxy group or arein the form of a reactive acid derivative thereof, for example in theform of a derived activated ester or reactive anhydride, or in the formof a reactive cyclic amide. The reactive acid derivatives may also beformed in situ.

Activated esters of compounds of formula VI having a terminal carboxygroup are especially esters unsaturated at the carbon atom linking theradical to be esterified, for example esters of the vinyl ester type,such as vinyl esters (obtainable, for example, by transesterification ofa corresponding ester with vinyl acetate; activated vinyl ester method),carbamoyl esters (obtainable, for example, by treatment of thecorresponding acid with an isoxazolium reagent; 1,2-oxazolium orWoodward method), or 1-lower alkoxyvinyl esters (obtainable, forexample, by treatment of the corresponding acid with a loweralkoxyacetylene; ethoxyacetylene method), or esters of the amidino type,such as N,N′-disubstituted amidino esters (obtainable, for example, bytreatment of the corresponding acid with a suitable N,N′-di-substitutedcarbodiimide, for example N,N′-dicyclohexylcarbodiimide or especiallyN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; carbodiimide method), orN,N-disubstituted amidino esters (obtainable, for example, by treatmentof the corresponding acid with an N,N-disubstituted cyanamide; cyanamidemethod), suitable aryl esters, especially phenyl esters suitablysubstituted by electron-attracting substituents (obtainable, forexample, by treatment of the corresponding acid with a suitablysubstituted phenol, for example 4-nitro-phenol, 4-methylsulfonylphenol,2,4,5-trichlorophenol, 2,3,4,5,6-pentachlorophenol or4-phenyldiazophenol, in the presence of a condensation agent, such asN,N′-dicyclohexyl-carbodiimide; activated aryl esters method),cyanomethyl esters (obtainable, for example, by treatment of thecorresponding acid with chloroacetonitrile in the presence of a base;cyanomethyl esters method), thio esters, especially unsubstituted orsubstituted, for example nitro-substituted, phenylthio esters(obtainable, for example, by treatment of the corresponding acid withunsubstituted or substituted, for example nitro-substituted,thio-phenols, inter alia by the anhydride or carbodiimide method;activated thiol esters method), or especially amino or amido esters(obtainable, for example, by treatment of the corresponding acid with anN-hydroxyamino or N-hydroxyamido compound, for exampleN-hydroxysuccinimide, N-hydroxypiperidine, N-hydroxyphthalimide,N-hydroxy-5-norbornene-2,3-dicarboxylicacid imide,1-hydroxybenzotriazole or3-hydroxy-3,4-dihydro-1,2,3-benzo-triazin-4-one, for example by theanhydride or carbodiimide method; activated N-hydroxy esters method).Internal esters, for example γ-lactones, can also be used.

Anhydrides of acids may be symmetric or preferably mixed anhydrides ofthose acids, for example anhydrides with inorganic acids, such as acidhalides, especially acid chlorides (obtainable, for example, bytreatment of the corresponding acid with thionyl chloride, phosphoruspentachloride, phosgene or oxalyl chloride; acid chloride method),azides (obtainable, for example, from a corresponding acid ester via thecorresponding hydrazide and treatment thereof with nitrous acid; azidemethod), anhydrides with carbonic acid semi-esters, for example carbonicacid lower alkyl semesters (especially chloroformic acid methyl esters)(obtainable, for example, by treatment of the corresponding acid withchloroformic acid lower alkyl esters or with a 1-loweralkoxycarbonyl-2-lower alkoxy-1,2-dihydroquinoline; mixedO-alkylcarbonic acid anhydrides method), or anhydrides withdihalogenated, especially dichlorinated, phosphoric acid (obtainable,for example, by treatment of the corresponding acid with phosphorusoxychloride; phosphorus oxychloride method), anhydrides with otherphosphoric acid derivatives (for example those obtainable withphenyl-N-phenyl-phosphoramidochloridate or by reaction ofalkylphosphoric acid amides in the presence of sulfonic acid anhydridesand/or racemisation-reducing additives, such as N-hydroxybenzotriazole,or in the presence of cyanophQsphonic acid diethyl ester) or withphosphorous acid derivatives, or anhydrides with organic acids, such asmixed anhydrides with organic carboxylic acids (obtainable, for example,by treatment of the corresponding acid with an unsubstituted orsubstituted lower alkane- or phenyl-lower alkane-carboxylic acid halide,for example phenylacetic acid chloride, pivalic acid chloride ortrifluoroacetic acid chloride; mixed carboxylic acid anhydrides method)or with organic sulfonic acids (obtainable, for example, by treatment ofa salt, such as an alkali metal salt, of the corresponding acid with asuitable organic sulfonic acid halide, such as a lower alkane- or aryl-,for example methane- or p-toluene-sulfonic acid chloride; mixed sulfonicacid anhydrides method) and symmetric anhydrides (obtainable, forexample, by condensation of the corresponding acid in the presence of acarbodiimide or 1-diethylaminopropyne; symmetric anhydrides method).

Suitable cyclic amides are especially amides with five-membereddiazacycles of aromatic character, such as amides with imidazoles, forexample imidazole (obtainable, for example, by treatment of thecorresponding acid with N,N′-carbonyldiimidazole; imidazole method), orpyrazole, for example 3,5-dimethylpyrazole (obtainable, for example, viathe acid hydrazide by treatment with acetylacetone; pyrazolide method).

As mentioned, derivatives of carboxylic acids used as acylating agentsmay also be formed in situ. For example, N,N′-disubstituted amidinoesters may be formed in situ by reacting a mixture of the startingmaterial of formula V and the acid used as acylating agent in thepresence of a suitable N,N′-disubstituted carbodiimide, for exampleN,N′-cyclohexylcarbodiimide or especiallyN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide. In addition, amino oramido esters of the acids used as acylating agents may be formed in thepresence of the starting material of formula V to be acylated, byreacting a mixture of the corresponding acid and amino startingmaterials in the presence of an N,N′-disubstituted carbodiimide, forexample N,N′-dicyclohexylcarbodiimide, and of an N-hydroxyamine orN-hydroxyamide, for example N-hydroxysuccinimide, where appropriate inthe presence of a suitable base, for example 4-dimethylamino-pyridine.Furthermore, activation in situ can be achieved by reaction withN,N,N′,N′-tetraalkyluronium compounds, such asO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate,O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetra-methyluroniumtetrafluoroborate (in the presence or absence of1,8-diazabicyclo[5.4.0]-undec-7-ene(1,5-5)) orO-(3,4-dihydro-4-oxo-1,2,3-benzotriazolin-3-yl)-N,N,N′,N′-tetra-methyluroniumtetrafluoroborate. Finally, phosphoric acid anhydrides of the carboxylicacids of formula VI can be prepared in siftu by reacting analkylphosphoric acid amide, such as hexamethylphosphoric acid triamide,in the presence of a sulfonic acid anhydride, such as 4-toluenesulfonicacid anhydride, with a salt, such as a tetrafluoroborate, for examplesodium tetrafluoroborate, or with another derivative ofhexamethylphosphoric acid triamide, such asbenzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluoride,preferably in the presence of a racemisation-reducing additive, such asN-hydroxybenzotriazole.

The amino group of compounds of formula V that participates in thereaction preferably carries at least one reactive hydrogen atom,especially when the carboxy, sulfonyl or phosphoryl group reactingtherewith is present in reactive form; it may, however, itself have beenderivatised, for example by reaction with a phosphite, such asdiethylchlorophosphite, 1,2-phenylene chlorophosphite,ethyldichlorophosphite, ethylene chlorophosphite ortetra-ethylpyrophosphite. A derivative of such a compound having anamino group is, for example, also a carbamic acid halide or anisocyanate, the amino group that participates in the reaction beingsubstituted by halocarbonyl, for example chlorocarbonyl, or modified inthe form of an isocyanate group, respectively.

Condensation to form an amide bond can be carried out in a manner knownper se, for example as described in standard works, such as Houben-Weyl,“Methoden der organischen Chemie”, 4th edition, Volume 15/II (1974),Volume IX (1955), Volume E11 (1985), Georg Thieme Verlag, Stuttgart,“The Peptides” (E. Gross and J. Meienhofer, eds.), Volumes 1 and 2,Academic Press, London and New York, 1979/1980, or M.Bodansky,“Principles of Peptide Synthesis”, Springer-Verlag, Berlin 1984.

The condensation of a free carboxylic acid with the appropriate aminecan be carried out preferably in the presence of one of the customarycondensation agents, or using carboxylic acid anhydrides or carboxylicacid halides, such as chlorides, or activated carboxylic acid esters,such as p-nitrophenyl esters. Customary condensation agents are, forexample, carbodiimides, for example diethyl-, dipropyl- ordicyclohexyl-carbodiimide or especiallyN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, also suitable carbonylcompounds, for example carbonylimidazole, 1,2-oxazolium compounds, forexample 2-ethyl-5-phenyl-1,2-oxazolium-3′-sulfonate and2-tert-butyl-5-methylisoxazolium perchlorate, or a suitable acylaminocompound, for example 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline,N,N,N,N′,N′-tetraaakyluronium compounds, such asO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate orespecially O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (in the presence or absence of1,8-diazabicyclo[5.4.0]undec-7-ene-(1,5-5)), also activated phosphoricacid derivatives, for example diphenylphosphorylazide,diethylphosphorylcyanide, phenyl-N-phenylphosphoro-amidochloridate,bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride or1-benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate.

If desired, an organic base is added, preferably a tertiary amine, forexample a tri-lower alkylamine, especially ethyldiisopropylamine or moreespecially triethylamine, and/or a heterocyclic base, for example4-dimethylaminopyridine or preferably N-methylmorpholine or pyridine.

The condensation of activated esters, reactive anhydrides or reactivecyclic amides with the corresponding amines is customarily carried outin the presence of an organic base, for example simple tri-loweralkylamines, for example triethylamine or tributylamine, or one of theabove-mentioned organic bases. If desired, a condensation agent isadditionally used, for example as described for free carboxylic acids.

The condensation of acid anhydrides with amines can be effected, forexample, in the presence of inorganic carbonates, for example ammoniumor alkali metal carbonates or hydrogen carbonates, such as sodium orpotassium carbonate or hydrogen carbonate (if desired together with asulfate).

Carboxylic acid chlorides, for example the chlorocarbonic acidderivatives derived from the acid of formula VI, are condensed with thecorresponding amines preferably in the presence of an organic amine, forexample the above-mentioned tri-lower alkylamines or heterocyclic bases,where appropriate in the presence of a hydrogen sulfate or a hydroxide,preferably an alkali metal hydroxide, such as sodium hydroxide.

The condensation is preferably carried out in an inert, aprotic,preferably anhydrous, solvent or solvent mixture, for example in acarboxylic acid amide, for example formamide or dimethylformamide, ahalogenated hydrocarbon, for example methylene chloride, carbontetrachloride or chlorobenzene, a ketone, for example acetone, a cyclicether, for example tetrahydrofuran or dioxane, an ester, for exampleethyl acetate, or a nitrile, for example acetonitrile, or in a mixturethereof, as appropriate at reduced or elevated temperature, for examplein a temperature range of from approximately −40° to approximately +100°C., preferably from approximately −10° to approximately +70° C., andwhen arylsulfonyl esters are used also at approximately from +100° to+200° C., especially at temperatures of from 10° to 30° C., and ifnecessary under an inert gas atmosphere, for example a nitrogen or argonatmosphere.

Aqueous, for example alcoholic, solvents, for example ethanol, oraromatic solvents, for example benzene or toluene, may also be used.When alkali metal hydroxides are present as bases, acetone may also beadded where appropriate.

The condensation can also be carried out in accordance with thetechnique known as solid-phase synthesis which originates from R.Merrifield and is described, for example, in Angew. Chem 97, 801-812(1985), Naturwissenschaften 71, 252-258 (1984) or in R. A. Houghten,Proc. Natl. Acad. Sci. USA 82, 5131-5135 (1985).

The freeing of protected groups may be effected in accordance with themethods described below under the heading “Removal of protectinggroups.”

Process c) (Formation of an amide bond)

In starting materials of formulae VII and VIII, functional groups, withthe exception of groups that are to participate in the reaction or thatdo not react under the reaction conditions, are protected independentlyof one another by one of the protecting groups mentioned under Processa).

The process is entirely analogous to that given under Process b) butcompounds of formula VII are used instead of those of formula V andcompounds of formula VIII are used instead of those of formula VI.

The freeing of protected groups may be effected in accordance with themethods described below under the heading “Removal of protectinggroups”.

Process d) (Formation of an amide bond)

In starting materials of formula IX and in the acid of formula VIIIasuitable for the introduction of tne identical acyl radicals, or inreactive derivatives thereof, functional groups that are not toparticipate in the reaction or that do not react under the reactionconditions, are protected independently of one another by one of theprotecting groups mentioned under Process a).

Preferred starting compounds of formula IX, which may be protected byprotecting groups, are those of formula II described below in thesection relating to starting compounds.

The process is entirely analogous to that given under Process b) butcompounds of formula IX are used instead of those of formula V andcompounds of formula VIIIa are used instead of those of formula VI.

The freeing of protected groups may be effected in accordance with themethods described below under the heading “Removal of protectinggroups”.

Process e) (Alkylation of a secondary nitrogen atom)

In starting materials of formula I′ and formula X or in reactivederivatives thereof, functional groups that are not to participate inthe reaction or that do not react under the reaction conditions, areprotected independently of one another by one of the protecting groupsmentioned under Process a).

A leaving group X is especially a nucleofugal leaving group selectedfrom hydroxy esterified by a strong inorganic or organic acid, such ashydroxy esterified by a mineral acid, for example a hydrohalic acid,such as hydrochloric, hydrobromic or hydriodic acid, hydroxy esterifiedby a strong organic sulfonic acid, such as a lower alkanesulfonic acidthat is unsubstituted or substituted, for example, by halogen, such asfluorine, or by an aromatic sulfonic acid, for example benzenesulfonicacid that is unsubstituted or substituted by lower alkyl, such asmethyl, halogen, such as bromine, and/or by nitro, for example amethanesulfonic, p-bromotoluenesulfonic or p-toluenesulfonic acid, andhydroxy esterified by hydrazoic acid.

The substitution can take place under the conditions of a first orsecond order nucleophilic substitution.

For example, one of the compounds of formula X wherein X is a leavinggroup having high polarisability of the electron shell, for exampleiodine, can be used in a polar aprotic solvent, for example acetone,acetonitrile, nitromethane, dimethyl sulfoxide or dimethylformamide. Thereaction can also be carried out in water, optionally in admixture withan organic solvent, for example ethanol, tetrahydrofuran or acetone, assolubiliser. The substitution reaction is carried out, as appropriate,at reduced or elevated temperature, for example in a temperature rangeof from approximately −40° to approximately 100° C., preferably fromapproximately −10° to approximately 50° C., and if necessary under aninert gas, for example under a nitrogen or argon atmosphere.

Process e) is not successful in all cases, is often possible only underspecial conditions and is therefore a less preferred process.

The freeing of protected groups may be effected in accordance with themethods described below under the heading “Removal of protectinggroups”.

Process f) (Reductive alkylation of a secondary amino group)

In starting materials of formula I′ and formula X* or in reactivederivatives thereof, functional groups that are not to participate inthe reaction or that do not react under the reaction conditions, areprotected independently of one another by one of the protecting groupsmentioned under Process a).

Reactive derivatives of the compounds of formula I are, for example,corresponding bisulfite adducts or especially semiacetals or ketals ofcompounds of formula X* with alcohols, for example lower alkanols; orthioacetals of compounds of formula X* with mercaptans, for examplelower alkanesulfides. The free aldehydes of formula X* are preferred.The reductive alkylation is preferably carried out with hydrogenation inthe presence of a catalyst, especially a noble metal catalyst, such asplatinum or especially palladium, which is preferably bonded to acarrier material, such as carbon, or a heavy metal catalyst, such asRaney nickel, at normal pressure or at pressures of from 0.1 to 10MegaPascal (MPa), or with reduction by means of complex hydrides, suchas borohydrides, especially alkali metal cyanoborohydrides, for examplesodium cyanoborohydride, in the presence of a suitable acid, preferablyrelatively weak acids, such as lower alkanecarboxylic acids orespecially a sulfonic acid, such as p-toluenesulfonic acid; in customarysolvents, for example alcohols, such as methanol or ethanol, or ethers,for example cyclic ethers, such as tetrahydrofuran, in the presence orabsence of water.

The freeing of protected groups may be effected in accordance with themethods described below under the heading “Removal of protectinggroups”.

Removal of Protecting Groups

The removal of protecting groups that are not constituents of thedesired end product of formula I, for example carboxy-, amino- andhydroxy-protecting groups, is effected in a manner known per se, forexample by means of solvolysis, especially hydrolysis, alcoholysis oracidolysis, or by means of reduction, especially hydrogenolysis orchemical reduction, and also photolysis, stepwise or simultaneously asappropriate, it being possible also to use enzymatic methods. Theremoval of the protecting groups is described, for example, in thestandard works mentioned hereinabove in the section relating toprotecting groups.

For example, protected carboxy, for example tert-lower alkoxycarbonyl,lower alkoxycarbonyl substituted in the 2-position by a trisubstitutedsilyl group or in the 1-position by lower alkoxy or by lower alkylthio,or unsubstituted or substituted diphenylmethoxycarbonyl can be convertedinto free carboxy by treatment with a suitable acid, such as formicacid, hydrogen chloride or trifluoroacetic acid, where appropriate withthe addition of a nucleophilic compound, such as phenol or anisole.Carboxy can be freed from lower alkoxycarbonyl also by bases, such ashydroxides, for example alkali metal hydroxides, such as NaOH or KOH.Unsubstituted or substituted benzyloxycarbonyl can be cleaved, forexample. by means of hydrogenolysis, i.e. by treatment with hydrogen inthe presence of a metal hydrogenation catalyst, such as a palladiumcatalyst. In addition, suitably substituted benzyloxycarbonyl, such as4-nitrobenzyloxycarbonyl, can be converted into free carboxy also byreduction, for example by treatment with an alkali metal dithionite,such as sodium dithionite, or with a reducing metal, for example zinc,or a reducing metal salt, such as a chromium(II) salt, for examplechromium(II) chloride, customarily in the presence of ahydrogen-yielding agent that, together with the metal, is capable ofproducing nascent hydrogen, such as an acid, especially a suitablecarboxylic acid, such as an unsubstituted or substituted, for examplehydroxy-substituted, lower alkanecarboxylic acid, for example aceticacid, formic acid, glycolic acid, diphenylglycolic acid, lactic acid,mandelic acid, 4-chloromandelic acid or tartaric acid, or in thepresence of an alcohol or thiol, water preferably being added. Bytreatment with a reducing metal or metal salt, as described above,2-halo-lower alkoxycarbonyl (where appropriate after conversion of a2-bromo-lower alkoxycarbonyl group into a corresponding 2-iodo-loweralkoxycarbonyl group) or aroylmethoxycarbonyl can also be converted intofree carboxy. Aroylmethoxycarbonyl can be cleaved also by treatment witha nucleophilic, preferably salt-forming, reagent, such as sodiumthiophenolate or sodium iodide. 2-(Tri-substituted silyl)-loweralkoxycarbonyl, such as 2-tri-lower alkylsilyl-lower alkoxycarbonyl, canalso be converted into free carboxy by treatment with a salt ofhydrofluoric acid that yields the fluoride anion, such as an alkalimetal fluoride, for example sodium or potassium fluoride, whereappropriate in the presence of a macrocyclic polyether (“crown ether”),or with a fluoride of an organic quaternary base, such as tetra-loweralkylammonium fluoride or tri-lower alkylaryl-lower alkylammoniumfluoride, for example tetraethylammonium fluoride or tetrabutylammoniumfluoride, in the presence of an aprotic, polar solvent, such as dimethylsulfoxide or N,N-dimethylacetamide. Carboxy protected in the form oforganic silyloxycarbonyl, such as tri-lower alkylsilyloxycarbonyl, forexample trimethylsilyloxycarbonyl, can be freed in customary manner bysolvolysis, for example by treatment with water, an alcohol or an acid,or, furthermore, a fluoride, as described above. Esterified carboxy canalso be cleaved enzymatically, for example by means of esterases orsuitable peptidases, for example using trypsin.

A protected amino group is freed in a manner known per se and, accordingto the nature of the protecting groups, in various ways, preferably bysolvolysis or reduction. Lower alkoxycarbonylamino, such astert-butoxycarbonylamino, can be cleaved in the presence of acids, forexample mineral acids, for example a hydrogen halide, such as hydrogenchloride or hydrogen bromide, or sulfuric or phosphoric acid, butpreferably hydrogen chloride, or in the presence of strong organicacids, such as a trihaloacetic acid, for example trifluoroacetic acid,or formic acid, in the presence or absence of polar solvents, such aswater, or ethers, preferably cyclic ethers, such as dioxane; ornitrites, such as acetonitrile, 2-halo-lower alkoxycarbonylamino (whereappropriate after conversion of a 2-bromo-lower alkoxycarbonylaminogroup into a 2-iodo-lower alkoxycarbonylamino group), or, dissolveddirectly in a liquid organic carboxylic acid, such as formic acid,aroylmethoxycarbonylamino or 4-nitrobenzyloxycarbonylamino can becleaved, for example, by treatment with a suitable reducing agent, suchas zinc in the presence of a suitable carboxylic acid, such as aqueousacetic acid. Aroylmethoxycarbonylamino can be cleaved also by treatmentwith a nucleophilic, preferably salt-forming, reagent, such as sodiumthiophenolate, and 4-nitrobenzyloxycarbonylamino also by treatment withan alkali metal dithionite, for example sodium dithionite. Unsubstitutedor substituted diphenylmethoxycarbonylamino, tert-loweralkoxycarbonylamino or 2-(tri-substituted silyl)-loweralkoxycarbonylamino, such as 2-tri-lower alkylsilyl-loweralkoxycarbonylamino, can be cleaved by treatment with a suitable acid,for example formic acid or trifluoroacetic acid; unsubstituted orsubstituted benzyloxycarbonylamino can be cleaved, for example, by meansof hydrogenolysis, i.e. by treatment with hydrogen in the presence of asuitable hydrogenation catalyst, such as a platinum or palladiumcatalyst; unsubstituted or substituted triarylmethylamino or formylaminocan be cleaved, for example, by treatment with an acid, such as amineral acid, for example hydrochloric acid, or an organic acid, forexample formic, acetic or trifluoroacetic acid, where appropriate in thepresence of water, and an amino group protected in the form ofsilylamino can be freed, for example, by means of hydrolysis oralcoholysis. An amino group protected by 2-haloacetyl, for example2-chloroacetyl, can be freed by treatment with thiourea in the presenceof a base, or with a thiolate salt, such as an alkali metal thiolate ofthiourea, and subsequent solvolysis; such as. alcoholysis or hydrolysis,of the resulting substitution product. Amino is freed fromtrifluoroacetylamino, for example, by hydrogenolysis with bases, such asalkali metal hydroxides or carbonates, such as Na₂CO₃ or K₂CO₃, in polarsolvents, for example alcohols, such as methanol, in the presence orabsence of water, at temperatures of from 0° to 100° C., especially atreflux temperature. An amino group protected by 2-(tri-substitutedsilyl)-lower alkoxycarbonyl, such as 2-tri-lower alkylsilyl-loweralkoxycarbonyl, can be converted into the free amino group also bytreatment with a salt of hydrofluoric acid that yields fluoride anions,as indicated above in connection with the freeing of a correspondinglyprotected carboxy group. A 1-aryl-lower alkylmethyl protecting groupwherein the lower alkyl radical is preferably branched in the1-position, such as 1-methyl-1-phenyl-ethyl, can be removed especiallyin the presence of a strong acid, such as sulfuric acid (e.g. 80%sulfuric acid) in aqueous solution, at preferred temperatures of from−10° to 30° C., especially at approximately 0° C.

Likewise, silyl, such as trimethylsilyl, bonded directly to a heteroatom, such as nitrogen, can be removed using fluoride ions.

Amino protected in the form of an azido group is converted into freeamino, for example, by reduction, for example by catalytic hydrogenationwith hydrogen in the presence of a hydrogenation catalyst, such asplatinum oxide, palladium or Raney nickel, by reduction using mercaptocompounds, such as dithiothreitol or mercaptoethanol, or by treatmentwith zinc in the presence of an acid, such as acetic acid. The catalytichydrogenation is preferably carried out in an inert solvent, such as ahalogenated hydrocarbon, for example methylene chloride, or in water orin a mixture of water and an organic solvent, such as an alcohol ordioxane, at approximately from 20° C. to 25° C., or with cooling orheating.

A hydroxy group protected by a suitable acyl group, by a tri-loweralkylsilyl group or by unsubstituted or substituted 1-phenyl-lower alkylis freed analogously to a correspondingly protected amino group. Ahydroxy group protected by 2,2-dichloroacetyl is freed, for example, bybasic hydrolysis, and a hydroxy group protected by tert-lower alkyl orby a 2-oxa- or 2-thia-aliphatic or -cycloaliphatic hydrocarbon radicalis freed by acidolysis, for example by treatment with a mineral acid ora strong carboxylic acid, for example trifluoroacetic acid. Adjacenthydroxy and amino groups that are protected together by a bivalentprotecting group, preferably, for example, by a methylene group mono- ordi-substituted by lower alkyl, such as by lower alkylidene, for exampleisopropylidene, cycloalkylidene, for example cyclohexylidene, orbenzylidene, can be freed by acid solvolysis, especially in the presenceof a mineral acid or a strong organic acid. A tri-lower alkylsilyl groupis likewise removed by acidolysis, for example by a mineral acid,preferably hydrofluoric acid, or a strong carboxylic acid. 2-Halo-loweralkoxycarbonyl is removed using the above-mentioned reducing agents, forexample a reducing metal, such as zinc, reducing metal salts, such aschromium(II) salts, or using sulfur compounds, for example sodiumdithionite or especially sodium sulfide and carbon disulfide.

When several protected functional groups are present, if desired theprotecting groups can be so selected that more than one such group canbe removed simultaneously, for example by removal of trifluoroacetyl asamino-protecting group by base catalysis, for example with K₂CO₃ inmethanol/water, and later removal of tert-butoxycarbonyl asamino-protecting group, for example with HCl in dioxane or acetonitrile(in the presence or absence of water) or with formic acid, or selectiveremoval of 1-methyl-1-phenyl-ethyl as amino-protecting group usingsulfuric acid; or generally by acidolysis, such as by treatment withtrifluoroacetic acid, or with hydrogen and a hydrogenation catalyst,such as a palladium-on-carbon catalyst. Conversely, the groups can alsobe so selected that they cannot all be removed simultaneously, butrather in a desired sequence, the corresponding intermediates beingobtained.

Additional Process Steps

In the additional process steps, which are optional, functional groupsof the starting compounds that are not to participate in the reactionmay be unprotected or may be in protected form, for example they may beprotected by one or more of the protecting groups mentioned above underProcess a). The protecting groups may be retained in the end products orsome or all of them may be removed in accordance with one of the methodsmentioned under the heading “Removal of protecting groups”.

Salts of compounds of formula I having a salt-forming group can beprepared in a manner known per se. For example, acid addition salts ofcompounds of formula I can be obtained, for example, by treatment withan acid or a suitable anion exchange reagent.

Salts can be converted into the free compounds in customary manner, forexample by treatment with a suitable basic agent.

Stereoisomeric mixtures, for example mixtures of diastereoisomers, canbe separated into the corresponding isomers in a manner known per se bysuitable separating procedures. For example, mixtures ofdiastereoisomers can be separated into the individual diastereo-isomersby fractional crystallisation, chromatography, solvent partitioning andthe like. Such separation can be carried out either at the stage of oneof the starting materials or with the compounds of formula I themselves.

In a compound of formula I wherein R₂ is phenyl, that phenyl radical canbe hydrogenated, for example by catalytic hydrogenation, especially inthe presence of heavy metal oxides, such as rhodium/platinum mixedoxides, for example with the Nishimura catalyst, preferably in a polarsolvent, such as an alcohol, for example methanol or ethanol, attemperatures of from 0° to 80° C., especially from 10° to 40° C., and ata preferred hydrogen pressure of from 1 to 10 atm, preferably at aboutnormal pressure.

In a compound of formula I wherein R₄ is 4-tetrazol-5-ylphenyl, a loweralkyl group, for example methyl, can be converted by reaction with alower alkyl halide or a lower alkylarylsulfonate, such as a lower alkyliodide or a lower alkyltoluenesulfonate, for example methyl iodide ortert-butyl iodide, preferably in the presence of caesium carbonate in amixture of a cyclic ether, such as dioxane, and an N,N-di-loweralkyl-lower alkanecarboxylic acid amide, such as dimethylformamide, atpreferred temperatures of from −10° to 40° C., especially from 0° toabout 30° C.

In a compound of formula I wherein R₄ is 4-(1- or 2-phenyl-lower alkyl,such as 1- or 2-(1-methyl-1-phenylethyl)-tetrazol-5-yl)phenyl, thephenyl-lower alkyl radical (preferably 1-methyl-1-phenylethyl) can beremoved by treatment with a strong mineral acid, such as sulfuric acid,in aqueous solution, preferably at temperatures of from −20° to 30° C.,for example at 0° C.

General Process Conditions

All the process steps given in this text can be carried out underreaction conditions known per se, but preferably under thosespecifically mentioned, in the absence or usually in the presence ofsolvents or diluents, preferably those solvents or diluents that areinert towards the reagents used and are solvents therefor, in theabsence or presence of catalysts, condensation agents or neutralisingagents, for example ion exchangers, such as cation exchangers, forexample in the H⁺ form, depending upon the nature of the reaction and/orthe reactants at reduced, normal or elevated temperature, for example ina temperature range of from approximately −100° to approximately 190°C., preferably from approximately −80° to approximately 150° C., forexample from −80° to −60° C., at room temperature, at from −20° to 40°C. or at the boiling point of the solvent used, under atmosphericpressure or in a closed vessel, optionally under pressure, and/or in aninert atmosphere, for example under an argon or nitrogen atmosphere.

In the case of all starting materials and intermediates, salts may bepresent when salt-forming groups are present. Salts may also be presentduring the reaction of such compounds, provided that the reaction willnot be affected.

In all reaction steps, any isomeric mixtures that are formed can beseparated into the individual isomers, for example diastereoisomers orenantiomers, or into any desired mixtures of isomers, for exampleracemates or diastereoisomeric mixtures, for example analogously to themethods described under the heading “Additional process steps”.

In certain cases, for example in the case of hydrogenation, it ispossible to carry out stereo-selective reactions so that, for example,individual isomers may be obtained more easily.

The solvents from which those suitable for a particular reaction can beselected include, for example, water, esters, such as lower alkyl-loweralkanoates, for example diethyl acetate, ethers, such as aliphaticethers, for example diethyl ether, or cyclic ethers, for exampletetrahydrofuran, liquid aromatic hydrocarbons, such as benzene ortoluene, alcohols, such as methanol, ethanol or 1- or 2-propanol,nitrites, such as acetonitrile, halogenated hydrocarbons, such asmethylene chloride, acid amides, such as dimethylformamide, bases, suchas heterocyclic nitrogen bases, for example pyridine, carboxylic acidanhydrides, such as lower alkanoic acid anhydrides, for example aceticanhydride, cyclic, linear or branched hydrocarbons, such as cyclohexane,hexane or isopentane, or mixtures of those solvents, for example aqueoussolutions, unless the description of the processes indicates otherwise.Such solvent mixtures can also be used in working-up, for example bychromatography or partitioning.

The invention relates also to those forms of the process in which acompound obtainable as intermediate at any stage is used as startingmaterial and the remaining steps are carried out or the process isinterrupted at any stage or a starting material is formed under thereaction conditions or is used ii the form of a reactive derivative orsalt, or a compound obtainable in accordance with the process of theinvention is produced under the process conditions and further processedin situ, it being preferable to use those starting materials whichresult in the compounds described above as being preferred, especiallythose described as being especially preferred, more especially preferredand/or very especially preferred.

The preparation of compounds of formula I is preferably carried outanalogously to the processes and process steps given in the Examples.

The compounds of formula I, including their salts, may also be obtainedin the form of hydrates, or their crystals may include, for example, thesolvent used for crystallisation.

Pharmaceutical Compositions:

The invention relates also to pharmaceutical compositions comprisingcompounds of formula I*, which means especially a compound of theformula I, and most especially of formula Ia.

The pharmacologically acceptable compounds of the present invention maybe used, for example, in the preparation of pharmaceutical compositionsthat comprise an effective amount of the active ingredient together orin admixture with a significant amount of inorganic or organic, solid orliquid, pharmaceutically acceptable carriers.

The invention relates also to a pharmaceutical composition suitable foradministration to a warm-blooded animal, especially a human being, forthe treatment or prevention of a disease that is responsive toinhibition of a retroviral protease, especially a retroviral aspartateprotease, such as HIV-1 or HIV-II gag protease, for example a retroviraldisease, such as AIDS or its preliminary stages, comprising a compoundof formula I*, or a pharmaceutically acceptable salt thereof, in anamount effective in the inhibition of the retroviral protease, togetherwith at least one pharmaceutically acceptable carrier.

The pharmaceutical compositions according to the invention arecompositions for enteral, such as nasal, rectal or oral, or parenteral,such as intramuscular or intravenous, administration to warm-bloodedanimals (human beings and animals) that comprise an effective dose ofthe pharmacological active ingredient alone or together with asignificant amount of a pharmaceutically acceptable carrier. The dose ofthe active ingredient depends on the species of warm-blooded animal,body weight, age and individual condition, individual pharmacokineticdata, the disease to be treated and the mode of administration.

The invention relates also to a method of treating diseases caused byviruses, especially by retroviruses, especially AIDS or its preliminarystages, wherein a therapeutically effective amount of a compound offormula I* or a pharmaceutically acceptable salt thereof is administeredin a dose that is effective in the treatment of said disease especiallyto a warm-blooded animal, for example a human being, who on account ofone of the mentioned diseases, especially AIDS or its preliminarystages, requires such treatment. The preferred dose to be administeredto warm-blooded animals, for example human beings of approximately 70 kgbody weight, is from approximately 3 mg to approximately 3 g, preferablyfrom approximately 10 mg to approximately 1.5 g, for exampleapproximately from 50 mg to 1000 mg per person per day, dividedpreferably into 1 to 3 single doses which may, for example, be of thesame size. Usually, children receive half of the adult dose.

The pharmaceutical compositions comprise from approximately 1% toapproximately 95%, preferably from approximately 20% to approximately90%, active ingredient. Pharmaceutical compositions according to theinvention may be, for example, in unit dose form, such as in the form ofampoules, vials, suppositories, dragees, tablets or capsules.

The pharmaceutical compositions of the present invention are prepared ina manner known per se, for example by means of conventional dissolving,lyophilising, mixing, granulating or confectioning processes.

Solutions of the active ingredient, and also suspensions, and especiallyisotonic aqueous solutions or suspensions, are preferably used, it beingpossible, for example in the case of lyophilised compositions thatcomprise the active ingredient alone or together with a carrier, forexample mannitol, for such solutions or suspensions to be made up priorto use. The pharmaceutical compositions may be sterilised and/or maycomprise excipients, for example preservatives, stabilisers, wettingagents and/or emulsifiers, solubilisers, salts for regulating theosmotic pressure and/or buffers, or acids, for example citric acid, andare prepared in a manner known per se, for example by means ofconventional dissolving or lyophilising processes. The said solutions orsuspensions may comprise viscosity-increasing substances, such as sodiumcarboxymethylcellulose, carboxymethylcellulose,hydroxypropylmethylcellulose (e.g. cellulose HPM603), silica gel,dextran, polyvinylpyrrolidone or gelatin.

Suspensions in oil comprise as the oil component the vegetable,synthetic or semi-synthetic oils customary for injection purposes. Theremay be mentioned as such especially liquid fatty acid esters thatcontain as acid component a long-chained fatty acid having from 8 to 22,especially from 12 to 22, carbon atoms, for example lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, arachidic acid, behenic acid, orcorresponding unsaturated acids, for example oleic acid, elaidic acid,erucic acid, brassidic acid or linoleic acid, if desired with theaddition of antioxidants, for example vitamin E, β-carotene or3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of those fattyacid esters has a maximum of 6 carbon atoms and is a mono- orpoly-hydric, for example a mono-, di- or tri-hydric, alcohol, forexample methanol, ethanol, propanol, butanol or pentanol or the isomersthereof, but especially glycol and glycerol. The following examples offatty acid esters are therefore to be mentioned: ethyl oleate, isopropylmyristate, isopropyl palmitate, “Labrafil M 2375” (polyoxyethyleneglycerol trioleate, Gattefosse, Paris), “Miglyol 812” (triglyceride ofsaturated fatty acids with a chain length of C₈ to C₁₂, Hüls AG,Germany), but especially vegetable oils, such as cottonseed oil, almondoil, olive oil, castor oil, soybean oil and more especially groundnutoil and sesame oil.

The injection compositions are prepared in customary manner understerile conditions; the same applies also to introducing thecompositions into ampoules or vials and sealing the containers.

Pharmaceutical compositions for oral administration can be obtained bycombining the active ingredient with solid carriers, if desiredgranulating a resulting mixture, and processing the mixture, if desiredor necessary, after the addition of appropriate excipients, intotablets, dragee cores or capsules. It is also possible for the activeingredients to be incorporated into plastics carriers that allow theactive ingredients to diffuse or be released in measured amounts.

Suitable carriers are especially fillers, such as sugars, for examplelactose, saccharose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example tricalcium phosphate or calcium hydrogenphosphate, and also binders, such as starch pastes using, for example,corn, wheat, rice or potato starch, gelatin, tragacanth,methylcellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired,disintegrators, such as the above-mentioned starches, also carboxymethylstarch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a saltthereof, such as sodium alginate. Excipients are especially flowconditioners and lubricants, for example silicic acid, talc, stearicacid or salts thereof, such as magnesium or calcium stearate, and/orpolyethylene glycol. Dragée cores are provided with suitable, optionallyenteric, coatings, there being used inter alia concentrated sugarsolutions which may comprise gum arabic, talc, polyvinylpyrrolidone,polyethylene glycol and/or titanium dioxide, or coating solutions insuitable organic solvents, or, for the preparation of enteric coatings,solutions of suitable cellulose preparations, such as ethylcellulosephthalate or hydroxypropylmethylcellulose phthalate.

Capsules are hard gelatin capsules and also soft, sealed capsules madeof gelatin and a plasticiser, such as glycerol or sorbitol. The hardgelatin capsules may comprise the active ingredient in the form ofgranules, for example with fillers, such as lactose, binders, such asstarches, and/or glidants, such as talc or magnesium stearate, and ifdesired with stabilisers. In capsules the active ingredient ispreferably dissolved or suspended in suitable oily excipients, such asfatty oils, paraffin oil or liquid polyethylene glycols, it likewisebeing possible for stabilisers and/or antibacterial agents to be added.There may be mentioned as such oils especially liquid fatty acid estersthat contain as acid component a long-chained fatty acid, for examplehaving from 8 to 22, especially from 12 to 22, carbon atoms, for examplelauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmiticacid, margaric acid, stearic acid, arachidic acid, behenic acid, orcorresponding unsaturated acids, for example oleic acid, elaidic acid,erucic acid, brassidic acid or linoleic acid, if desired with theaddition of antioxidants, for example vitamin E, β-carotene or3,5-di-tert-butyl-4-hydroxy-toluene. The alcohol component of thosefatty acid esters has a maximum of 6 carbon atoms and is a mono- orpoly-hydric, for example a mono-, di- or tri-hydric, alcohol, forexample methanol, ethanol, propanol, butanol or pentanol or the isomersthereof, but especially ethylene or propylene glycol and glycerol. Thefollowing examples of fatty acid esters are therefore to be mentioned:efihyl oleate, isopropyl myristate, isopropyl palmitate, “Labrafil M2375” (polyoxyethylene glycerol trioleate, Gattefossé, Paris), “Miglyol812” (tri-glyceride of saturated fatty acids with a chain length of C₈to C₁₂, Hüls AG, Germany), but especially vegetable oils, such ascottonseed oil, almond oil, olive oil, castor oil, groundnut oil,soybean oil and more especially sesame oil. Paraffin oil is alsopossible. Stabilisers, such as emulsifiers, wetting agents orsurfactants, binders, such as starch pastes using, for example, corn,wheat, rice or potato starch, gelatin, tragacanth, methylcellulose,hydroxypropylmethylcellulose or hydroxypropylcellulose (preferred),sodium carboxymethylcellulose, cyclodextrin(s) and/orpolyvinylpyrrolidone, and/or antibacterial agents may be added. Suitableemulsifiers are especially oleic acid, non-ionic surfactants of thefatty acid polyhydroxy alcohol ester type, such as sorbitan monolaurate,monooleate, monostearate or monopalmitate, sorbitan tristearate ortrioleate, polyoxyethylene adducts of fatty acidpolyhydroxy alcoholesters, such as polyoxyethylene sorbitan monolaurate, mono-oleate,monostearate, monopalmitate, tristearate or trioleate, polyethyleneglycol fatty acid esters, such as polyoxyethyl stearate, polyoxyethyleneglycol (300 or 400) stearate, poly-ethylene glycol 2000 stearate,especially ethylene oxide/propylene oxide block polymers of the®Pluronic type (Wyandotte Chem. Corp.; trade mark of BASF, FRG) or®Synperonic type (ICI). For example, if the active ingredient is notsoluble in the mentioned oils it is present in the form of a suspension,for example having a particle size of approximately from 1 to 100 mm.Such suspensions may also be used as such, that is to say withoutcapsules.

Colourings or pigments may be added to the tablets or dragee coatings orto capsule walls, for example for identification purposes or to indicatedifferent doses of active ingredient.

Starting Materials:

The present invention relates also to novel starting materials and/orintermediates and to processes for their preparation. The startingmaterials used and the reaction conditions selected are preferably thosewhich result in the compounds described as being preferred.

In the preparation of all starting materials, free functional groupsthat are not to participate in the reaction in question may beunprotected or may be in protected form, for example they may beprotected by the protecting groups mentioned above under Process a).Those protecting groups can be removed at suitable times by thereactions described under the heading “Removal of protecting groups,”.

The starting materials of Process a) are known or, if novel, can beprepared in accordance with processes known per se, for example thecompounds of formula III can be prepared from hydrazine or suitablederivatives thereof, and the compounds of formula IV can be preparedfrom suitable amino acids or analogues thereof, for example having oneof the mentioned side chains R₃.

The compounds of formula III can be obtained, for example, fromcompounds of formula

H₂N—NH—R₇  (XI),

which are known per se or can be prepared from hydrazine by theintroduction of protecting groups as described under Process a) and inwhich R₇ is hydrogen or an amino-protecting group as described aboveunder Process b), especially tert-lower alkoxycarbonyl, such astert-butoxycarbonyl, aryl-lower alkoxycarbonyl, such asbenzyloxycarbonyl or 9-fluorenylmethoxycarbonyl, or one of theabove-mentioned acylamino-protecting groups, especially trifluoroacetyl,by alkylation with a compound of formula X as described above underProcess e), or by reaction of the radical of sub-formula

—R₄  (A)

wherein R₄ is as defined or compounds of formula I, by reaction of asuitable carbonyl compound of formula X*, or a reactive derivativethereof, both as defined under Process f), with the free amino group ofthe compound of formula XI or or an acylated derivative thereof andsubsequent reduction of the resulting hydrazone to form a hydrazinederivative of formula

the radicals in all the mentioned compounds being as defined above andfunctional groups in the reagents involved that are not to participatein the reaction being protected as necessary, and removal of theprotecting group R₇ as necessary and by condensation under theconditions mentioned above under Process b) with an acid of formula VI,or an acid derivative thereof mentioned under Process b).

The carbonyl compounds of formula X*, or reactive derivatives thereof,suitable for the introduction of the radical of sub-formula A that areused for the preparation of the compounds of formula XII, as definedabove under Process f), are aldehydes or reactive derivatives thereof,the reactive carbonyl group of which, after the reaction with compoundsof formula XI and the subsequent reduction, is a constituent of one ofthe mentioned radicals of sub-formula A.

The reaction of the carbonyl compounds with the compounds of formula XIto form the corresponding hydrazones is carried out under the conditionscustomarily used for the reaction of carbonyl compounds with amines,preferably in polar organic solvents, for example ethers, such astetrahydrofuran or diethyl ether, alcohols, such as methanol or ethanol,carboxylic acid amides, such as dimethylformamide, or esters, such asethyl acetate, or in aqueous solution, preferably in methanol, and alsoin the presence or absence of acid catalysts, for example carboxylicacids, such as formic acid or acetic acid, or sulfonic acids, such asp-toluenesulfonic acid, at temperatures of from 0° C. to the refluxtemperature of the reaction mixture, preferably at temperatures of from20° C. to the reflux temperature of the reaction mixture.

Compounds of formula

wherein R₄ and R₇ are as defined for compounds of formula XII areobtained.

The reduction of the resulting hydrazones of formula XII* is preferablycarried out by hydrogenation in the presence of a suitable catalyst orwith complex hydrides in the presence of acids. As catalysts suitablefor hydrogenation there are used metals, such as nickel, iron, cobalt orruthenium, or noble metals or oxides thereof, such as palladium orrhodium or oxides thereof, optionally, for example, applied to asuitable carrier, such as barium sulfate, aluminium oxide or carbon(active carbon) or in the form of skeleton catalysts, such as Raneynickel. Solvents customarily used for the catalytic hydrogenation are,for example, water, alcohols, such as methanol or ethanol, esters, suchas ethyl acetate, ethers, such as dioxane, chlorinated hydrocarbons,such as dichloromethane, carboxylic acid amides, such asdimethylformamide, or carboxylic acids, such as glacial acetic acid, ormixtures of those solvents. The hydrogenation is carried out preferablyat temperatures of from 10° to 250° C., especially from room temperatureto 100° C., and preferably at hydrogen pressures of from 1 to 200 bar,especially from 1 to 10 bar, in the customary apparatus. For thereduction with complex hydrides, especially borohydrides, such as alkalimetal cyanoborohydrides, for example sodium cyanoborohydride, it ispreferable to add weak acids, such as sulfonic acids, for examplep-toluenesulfonic acid, or carboxylic acids, such as acetic acid,preferably in alcohols, such as methanol or ethanol, or mixtures thereofwith water (see, for example, Tetrahedron 49, 8605-8628 (1993)).

It is also possible for compounds of formula XI to be alkylated byreduction directly with compounds of formula X*, or reactive derivativesthereof, as defined under Process f), under conditions analogous tothose mentioned in Process f).

Also especially preferred for the preparation of compounds of formula XIare reaction conditions analogous to those described in J. Chem. Soc.Perkin I, 1712 (1975).

Compounds of formula III can also be obtained, for example, by reactinga compound of formula XII*, as defined above, wherein R₇ is hydrogen(obtainable, for example, by the removal of protecting groups when R₇ isa protecting group), directly, with condensation under the conditionsmentioned above under Process b) with acids of formula VI, or the acidderivatives thereof mentioned under Process b), to form compounds offormula

wherein the radicals are as defined for compounds of formula I, whichare then converted into compounds of formula III by reduction underconditions analogous to the conditions mentioned for the reduction ofhydrazones of formula XII*.

Compounds to formula III* can also be obtained from the correspondingcompounds of formula III′, which are as defined as described below, byreacting the latter with compounds of formula X*, as defined above, toform the hydrazones of formula III* under conditions analogous to thosedescribed above for the reaction of carbonyl compounds of formula X*with hydrazines of formula XI.

A compound of formula IV can be obtained, for example, by reduction ofan amino acid of formula

wherein R₈ is hydrogen or especially one of the amino-protecting groupsmentioned under Process a), especially tert-lower alkoxycarbonyl, suchas tert-butoxycarbonyl, aryl-lower alkoxycarbonyl, such asbenzyloxycarbonyl or 9-fluorenylmethoxycarbonyl, or one of theacylamino-protecting groups mentioned under Process a), especiallytrifluoroacetyl, and R₃ is as defined for compounds of formula I, toform an aldehyde of formula

wherein the radicals are as last defined, subsequent reaction of thataldehyde with a ylid compound, preferably a sulfur ylid compound, toform an epoxide of formula

wherein the radicals are as last defined, removal of the protectinggroup R₈ (the resulting free amino compound wherein R₈=hydrogen may bestable, for example in the form of an acid addition salt) and finallyacylation of the amino group of the resulting compound with an acid offormula VIII, wherein the radicals are as defined for formula VIII,under suitable conditions analogous to the conditions described forProcess b).

The reduction of amino acids of formula XIII to the correspondingaldehydes of formula XIV is carried out, for example, by reduction tothe corresponding alcohols and subsequent oxidation to the mentionedaldehydes.

The reduction to the alcohols (a free compound or (if necessary afterthe introduction of protecting groups, as described under Process a)) acompound N-protected by R₈, having the formula

wherein the radicals are as defined for compounds of formula XIII) iscarried out, for example, by hydrogenation of the acid halides or otheractivated carboxylic acid derivatives mentioned under Process b) underthe conditions mentioned for the hydrogenation of hydrazones obtainedfrom compounds of formula XII, with diborane or with complex hydrides,such as sodium borohydride. The subsequent oxidation of the resultingalcohols is possible, for example, by oxidation of the hydroxy groupwith a sulfoxide, such as dimethyl sulfoxide, in the presence of areagent that activates the hydroxy group, such as a carboxylic acidchloride, for example oxalyl chloride, in inert solvents, for example ahalogenated hydrocarbon, such as dichloromethane, and/or an acyclic orcyclic ether, such as tetrahydrofuran, at from −80° to 0° C., forexample from −78° to −50° C., or by oxidation, for example, with chromicacid or a derivative thereof, such as pyridinium chromate or tert-butylchromate, dichromate/sulfuric acid, sulfur trioxide in the presence ofheterocyclic bases, such as pyridine/SO₃, and also nitric acid,pyrolusite or selenium dioxide, in water, organic solvents, such ashalogenated solvents, for example methylene chloride, carboxylic acidamides, such as dimethylformamide, or di-lower alkylsulfoxides, such asdimethyl sulfoxide, in the presence or absence of basic amines, forexample tri-lower alkylamines, such as triethylamine, at temperatures offrom −50° to 100° C., preferably at from −10° to 50° C., or by catalyticdehydrogenation, for example in the presence of metallic silver, copper,copper chromium oxide or zinc oxide at approximately from 200° to 400°C. (in the contact tube) with subsequent rapid cooling. Oxidation with2,2,6,6-tetramethyl-piperidin-1-oxyl in the presence of NaOCl is alsopossible (see Anelli et al., Org. Synth. 69, 212 (1990)).

The direct reduction of the amino acids to the aldehydes is alsopossible, for example by hydrogenation in the presence of a partiallypoisoned palladium catalyst or by reduction of the corresponding aminoacid esters, for example the lower alkyl esters, such as the ethylester, with complex hydrides, for example borohydrides, such as sodiumborohydride, or preferably aluminium hydrides, for example lithiumaluminium hydride, lithium tri(tert-butoxy)aluminium hydride orespecially diisobutylaluminium hydride, in non-polar solvents, forexample in hydrocarbons or aromatic solvents, such as toluene, at from−100° to 0° C., preferably from −70° to −30° C., and subsequent reactionto form the corresponding semicarbazones, for example with thecorresponding acid salts of semicarbazones, such as semicarbazidehydrochloride, in aqueous solvent systems, such as alcohol/water, forexample ethanol/water, at temperatures of from −20° to 60° C.,preferably from 10° to 30° C., and reaction of the resultingsemicarbazone with a reactive aldehyde, for example formaldehyde, in aninert solvent, for example a polar organic solvent, for example acarboxylic acid amide, such as dimethylformamide, at temperatures offrom −30° to 60° C., preferably from 0° to 30° C., and then with anacid, for example a strong mineral acid, such as a hydrogen halide, inaqueous solution, optionally in the presence of the solvent usedpreviously, at temperatures of from −40° to 50° C., preferably from −10°to 30° C. The corresponding esters are obtained by reaction of the aminoacids with the corresponding carboxylic acids, for example ethanol,analogously to the conditions employed in the condensation under Processb), for example by reaction with inorganic acid halides, such as thionylchloride, in organic solvent mixtures, such as mixtures of aromatic andalcoholic solvents, for example toluene and ethanol, at temperatures offrom −50° to 50° C., preferably from −10° to 20° C.

The preparation of the compounds of formula XIV is carried out in anespecially preferred manner under conditions analogous to the reactionconditions mentioned in J. Org. Chem. 47, 3016 (1982) or J. Org. Chem.43, 3624 (1978).

A sulfur ylid suitable for the conversion of compounds of formula XIVinto the epoxides of formula XV is. for example, a dialkylsulfoniummethylide, for example dimethylsulfonium methylide, an alkyl- orphenyl-dialkylaminosulfoxonium methylide, for example methyl- orphenyl-dimethylaminosulfoxonium methylide, or a dialkylsulfoxoniummethylide, for example dimethyl- or diethyl-sulfoxonium methylide.

The sulfur ylid compound in question is advantageously prepared in situfrom the corresponding sulfonium or sulfoxonium salt and a base, forexample sodium hydride, in a dipolar aprotic solvent, for exampledimethyl sulfoxide, or an ether, for example tetrahydrofuran or1,2-dimethoxyethane, and is then reacted with the compound of formulaXIV. The reaction is normally carried out at room temperature, withcooling, for example down to −20° C., or with gentle heating, forexample up to 40° C. The sulfide, sulfinamide or sulfoxide formed at thesame time is removed in the subsequent aqueous working-up.

The reaction with a sulfur ylid is effected in an especially preferredmanner analogously to the conditions mentioned in J. Org. Chem. 50, 4615(1985).

A compound of formula XV can also be obtained from a compound of formulaXIV, as defined above, by reaction thereof with a tri-loweralkylsilylmethyl Grignard compound, for example prepared from thecorresponding halomethylsilane, such as chloromethyl-trimethylsilane, inan inert solvent, for example an ether, such as dioxane or diethylether, at temperatures of from 0° to 50° C., for example from roomtemperature to approximately 40° C., subsequent elimination with removalof the silyl radical and formation of a double bond, for example bymeans of a Lewis acid, such as BF₃, any amino-protecting group R₈preferably also being removed, in an inert solvent, for example anether, such as diethyl ether, or a halogenated hydrocarbon, such asdichloromethane, or a mixture thereof, at temperatures of from −50° C.to the reflux temperature, especially from 0° to 30° C., if necessaryacylation again with the introduction of an aminoaprotecting group R₁₂,as defined above, and oxidation of the resulting double bond to form theoxirane, preferably with a percarboxylic acid, for examplem-chloroperbenzoic acid or monoperphthalic acid (for example inmagnesium salt form), in an inert solvent, for example a halogenatedhydrocarbon, such as dichloromethane, or alcohols, such as methanol,lower alkanoylnitriles, such as acetonitrile, water or mixtures thereof,at temperatures of from −20° C. to the reflux temperature of themixture, for example at from 10° to 50° C.

Compounds of formula IV are preferably prepared by starting directlywith an alcohol of formula XIII*, as defined above, which is alsocommercially available, reacting that alcohol with an acid of formulaVIII, or with a reactive derivative thereof, as defined for Process c),under the conditions mentioned therein, with, if necessary, protectinggroups being introduced, as described under Process a), and removed atsuitable times, as described under the heading “Removal of protectinggroups”, there being obtained a compound analogous to the compound offormula XIII* wherein the place of R₈ is taken by the corresponding acylradical from the acid of formula VIII; the resulting compound isoxidised under conditions analogous to those mentioned for the oxidationof alcohols of formula XIII* to form the corresponding aldehyde offormula

wherein the radicals are as defined, and that aldehyde is thenconverted, for example with an ylid compound, as described for theconversion of compounds of formula XIV into compounds of formula XV,into the compound of formula IV.

The starting materials of Processes b), c) and d) are known or, ifnovel, can be prepared in accordance with processes known per se: forexample a compound of formula V can be prepared from a suitablehydrazine derivative of formula XII wherein R₇ is a protecting group andthe remaining radicals are as defined for compounds of formula V and asuitable epoxide of formula IV wherein the radidhis are as defined forcompounds of formula I (Process b); a compound of formula VII can beprepared from a suitable hydrazine derivative of formula III wherein theradicals are as defined for compounds of formula I and a suitableepoxide of formula XV wherein R₈ is a protecting group and the remainingradicals are as defined for compounds of formula I (Process c); and thecompound of formula IX can be prepared from a suitable hydrazinederivative of formula XII wherein R₇ is hydrogen and the remainingradicals are as defined for compounds of formula I and a suitableepoxide of formula XV wherein R₈ is a protecting group and the remainingradicals are as defined for compounds of formula I (Process d),analogously to Process a), optionally using and removing protectinggroups, as described under Process a) and under the heading “Removal ofprotecting groups”, the protecting groups R₇ and R₈ preferably being asdefined above in the definition of compounds of formula XI and XIII,respectively.

Compounds of formula I′, wherein the substituents are as defined above,can be prepared, for example, from compounds of formula III′,

wherein the radicals are as defined for compounds of formula I, in amanner analogous to that described in Process b), by reaction with acompound of formula IV, wherein any functional groups present that arenot to participate in the reaction may be protected as described inProcess b) and freed again after the reaction.

Compounds of formula III′ can be obtained from compounds of formula XI,as defined above, by reaction with an acid of formula VI, or a reactiveacid derivative thereof, wherein the radicals are as defined above, in amanner analogous to that described for the reaction of compounds offormula XII with an acid of formula VI, and, as necessary, subsequentremoval of the protecting group R₇ in accordance with one of the methodsdescribed under the heading “Removal of protecting groups”.

Where two amino-protecting groups are present they may be identical ordifferent.

The amino-protecting groups used are, for example, the amino-protectinggroups mentioned above under Process a). Preference is given to thecorresponding compounds wherein the protecting groups are selected fromthose described as being preferred for R₇ and R₈ in compounds offormulae XI awid XIII, respectively.

The preparation of the protected compounds of formula I is carried out,for example, in accordance with any one of the processes mentionedhereinbefore, especially from compounds of formulae III and IV whereinfunctional groups may be protected by protecting groups, as describedunder Process a).

The acids of formulae VI, VIII and VIIIa and the compounds of formula X,and the aldehydes suitable for the introduction of the radical ofsub-formula A that are used for the preparation of the compounds offormula XII can be prepared in accordance with processes known per se ifthey are not already known.

The preparation of the acids of formula VI is effected by reaction ofderivatives of a lower alkoxycarboxylic acid that are suitable for theintroduction of lower alkoxycarbonyl radicals, for example by reactionof the corresponding pyrocarbonic acid di-lower alkyl esters (especiallypyrocarbonic acid dimethyl ester; Aldrich, Buchs, Switzerland) orpreferably haloformic acid lower alkyl esters, such as chloroformic acidlower alkyl esters (especially chloroformic acid methyl ester, Fluka,Buchs, Switzerland), with amino acids of the formula

wherein R₅ is as defined for compounds of formula VI, under conditionsanalogous to those described for acylation under Process b), especiallyin an aqueous alkali metal hydroxide solution, for example aqueoussodium hydroxide solution, in the presence of dioxane at temperatures offrom 20 to 100° C., especially from 50 to 70° C.

Correspondingly, the compounds of formula VIII can be obtained fromamino acids of formula

wherein R₂ is as defined for compounds of formula I, and the compoundsof formula VIIIa can be obtained from amino acids of formula

wherein R₂′ is as defined for compounds of formula VIII′, by reactionwith derivatives of a lower alkoxycarboxylic acid that are suitable forthe introduction of lower alkoxycarbonyl radicals.

The amino acids of formulae XVI, XVII and XVIII are known or can beprepared in accordance with processes known per se. They are preferablyin the (S)-form (in respect of the α-carbon atom).

Compounds of formula IV can also be prepared by condensing a compound offormula XIX

with a compound of formula XVIII, as defined above. The condensationwith an acid of formula VIII, or an acid derivative thereof, is carriedout under conditions analogous to those mentioned above under Processe). A compound of formula XX,

wherein R₁ and R₂ are as defined for compounds of formula I, isobtained.

Epoxidation with oxygen, or preferably chemically bonded oxygen, forexample in hydroperoxides, hydrogen peroxides or peroxy acids, such asperbenzoic acid, performic acid, peracetic acid, monoperoxyphthalicacid, pertungstic acid or especially m-chloroperbenzoic acid, in inertsolvents, such as ethers, for example diethyl ether, or chlorinatedhydrocarbons, such as chloroform or dichloromethane, at preferredtemperatures of from −20 to 50° C., yields a compound of formula IV, asdefined above.

The starting material of formula XIX is obtained preferably by reactionof a compound of formula XIV wherein R₃ is phenyl and R₈ is a protectinggroup with a Grignard reagent that introduces the methylidene group,especially with the trimethylsilylmethyl Grignard reagent(ClMgCH₂Si(CH₃)₃— which can be prepared from chloromethyltrimethylsilane(Fluka, Buchs, Switzerland) under conditions customary for thepreparation of Grignard compounds) in an inert solvent, such as anether, for example diethyl ether, at a preferred temperature of from −65to 0° C. and subsequent removal of the hydroxy group and thetrimethylsilyl group, for example with boron trifluoride in an ether,such as diethyl ether, at preferred temperatures of from −20 to 30° C.,with simultaneous removal of the protecting group R₈ (especially in thecase of removal of the tert-butoxycarbonyl protecting group) or withsubsequent removal of the protecting group, as described under theheading “Removal of protecting groups”.

Also possible is synthesis starting with a compound of formula XIVwherein R₃ is phenyl and R₈ is a protecting group using a suitableWittig reagent, such as methyltriphenylphosphonium bromide or iodide inthe presence of a strong base, such as sodium amide, at temperatures offrom −90 to 0° C., followed by removal of the protecting group R₈ inaccordance with the conditions mentioned under the heading “Removal ofprotecting groups”.

Compounds of formula X* are known, can be prepared in accordance withprocesses known per se or can be prepared, for example, as follows:

Using a compound of formula XXI,

wherein Hal is halogen, especially bromine or chlorine, and reacting itwith an unsaturated heterocycle that has from 5 to 8 ring atoms,contains from 1 to 4 hetero atoms selected from nitrogen, oxygen,sulfur, sulfinyl (—SO—) and sulfonyl (—SO₂—) and is unsubstituted orsubstituted by lower alkyl or by phenyl-lower alkyl, especially withthiazole or thiophene, in the presence oftetrakis(triphenylphosphine)palladium as catalyst and in the presence ofan alkali metal lower alkanoate, such as potassium acetate, in asuitable solvent, especially a N,N-di-lower alkyl-lower alkanoyl-amide,such as dimethyl acetamide, at preferred temperatures of from 80° C. tothe boiling temperature of the mixture, for example at approximately150° C., the corresponding compound of formula X*, especially4-(thiazol-5-yl)-benzaldehyde or 4-(thiopen-2-yl)-benzaldehyde, can beobtained.

Alternatively, it is possible, starting with a compound of formula XXI,as last defined, to obtain the corresponding di-lower alkylacetal (seefor example J. Org. Chem. 56, 4280 (1991)), for example thebromobenzaldehyde dimethylacetal (obtainable, for example, by reactionof 4-bromobenzaldehyde with orthoformic acid trimethyl ester in analcohol, such as methanol, in the presence of an acid, such asp-toluenesulfonic acid (can also be used in the form of the hydrate)).The resulting 4-halo-benzaldehyde di-lower alkylacetal is thenconverted, by reaction with magnesium in the presence of a catalyticamount of iodine in a suitable solvent, such as an ether, for exampletetrahydrofuran, at preferred temperatures of from 0° to 70° C., intothe corresponding Grignard reagent of formula XXII,

wherein Hal is halogen, especially chlorine or bromine, and Z is loweralkyl, which is then reacted, in the presence of1,3-bis(diphenylphosphino)propane nickel(II) chloride as catalyst in asuitable solvent, such as ether, for example tetrahydrofuran, therebeing added in an especially preferred process variant a suitablecomplex hydride, especially diisobutylaluminium hydride, (for exampledissolved in a hydrocarbon, such as hexane), at preferred temperaturesof from 0° to 60° C., with a compound of formula XXIII,

R₉-Hal′  (XXIII)

wherein R₉ is an unsaturated heterocycle that has from 5 to 8 ringatoms, contains from 1 to 4 hetero atoms selected from nitrogen, oxygen,sulfur, sulfinyl (—SO—) and sulfonyl (—SO₂—) and is unsubstituted orsubstituted by lower alkyl or by phenyl-lower alkyl, and wherein Hal′ ischlorine or especially bromine, with subsequent acid hydrolysis of theacetal (for example with hydrogen chloride in water), to form thecorresponding aldehyde compound of formula X*. Especially preferred ascompounds of formula XXIII are 2-bromothiazole, 2- or 3-bromopyridine or2-chloropyrazine in the preparation of the following compounds offormula X*: 4-(thiazol-2-yl)-benzaldehyde, 4-(pyridin-2-yl or-3-yl)-benzaldehyde or 4-(pyrazin-2-yl)-benzaldehyde.

Compounds of formula X* wherein R₄ is 4-(tetrazolyl-5-yl)-phenyl, areobtainable by reaction of 4-cyanobenzaldehyde with an alkali metalazide, such as sodium azide, in the presence of a suitable alkali metalhalide, such as lithium chloride, in a suitable solvent, such as2-methoxyethanol, preferably at boiling temperature. By reaction withphenyl-lower alkyl halides or preferably with phenyl-lower alkenes, suchas 2-phenylpropene, in a suitable solvent, such as toluene, and asuitable acid, such as methanesulfonic acid, preferably under reflux,the corresponding 1- or 2-phenyl-lower alkyl compounds of formula X* areobtained. By reaction with a lower alkyl halide, such as the iodide orbromide, for example methyl iodide, in the presence of alkali metalcarbonates, such as potassium or especially caesium carbonate, andsuitable solvents, such as dioxane, at preferred temperatures of fromapproximately 0° to approximately 30° C., compounds of formula X*substituted in the tetrazolyl ring by lower alkyl or by phenyl-loweralkyl, especially 4-(1-methyl-tetrazol-5-yl)-benzaldehyde, are obtained.

Compounds of formula X may be obtained from the corresponding compoundsof formula X* by reduction of the aldehyde function to a hydroxymethylgroup (for example with complex hydrides, such as lithium aluminiumhydride in ethanol, disiamylborane in tetrahydrofuran, sodiumborohydride in the presence of lithium chloride in diglycol or sodiumborohydride in ethanol) and subsequent introduction of the radical X byesterification by a strong inorganic or organic acid, such as by amineral acid, for example a hydrohalic acid, such as hydrochloric,hydrobromic or hydriodic acid, or by a strong organic sulfonic acid,such as an unsubstituted or substituted, for example halo-substituted,for example fluoro-substituted, lower alkanesulfonic acid or an aromaticsulfonic acid, for example a benzenesulforic acid that is unsubstitutedor substituted by lower alkyl, such as methyl, halogen, such as bromine,and/or by nitro, for example methanesulfonic acid,p-bromotoluenesulfonic acid or p-toluenesulfonic acid, or hydrazoicacid, in accordance with standard methods. For example, by reaction withinorganic acid halides, such as thionyl or phosphoryl halides (forexample the chlorides, bromides or iodides), halogen radicals X can beintroduced, or the remaining compounds of formula X can be obtained byreaction with other suitable organic or inorganic acids, such as strongorganic sulfonic acids (used for example as acid chlorides).

Starting materials (especially those of formulae IV*, V*, VII*, IX* and(I′)*) can also be prepared analogously to the processes mentioned in EP0 521 827 or EP 0 672 448 or are obtainable from the reference sourcesmentioned therein, or they are known, can be prepared according toprocesses known per se or are commercially available.

The preparation of starting materials for the preparation of compoundsof formula I is preferably carried out analogously to the processes andprocess steps mentioned in the Examples.

Of the starting materials according to the invention the following areespecially preferred (when radicals are not specifically defined, themeanings mentioned in the definition for compounds of formula I apply ineach case):

(1) compounds of formula XX wherein R₁ is methoxycarbonyl orethoxycarbonyl and R₂ is tert-butyl;

(2) compounds of formula IV wherein R₁ is methoxycarbonyl orethoxycarbonyl and R₂ is tert-butyl;

(3) compounds of formula III*, especially those wherein R₅ is tert-butyland R₆ is methoxy- or ethoxycarbonyl;

(4) compounds of formula XII;

(5) compounds of formula XII*;

(6) compounds of formula III;

(7) compounds of formula V;

(8) compounds of formula VII;

(9) compounds of formula IX;

(10) compounds of formula X;

(11) a compound of formula X* selected from4-(1-methyl-tetrazol-5-yl)-benzaldehyde, 4-(thiazol-2-yl)-benzaldehyde,4-(pyridin-2-yl or -3-yl)-benzaldehyde, 4-(pyrazin-2-yl)-benz-aldehyde,4-(thiazol-5-yl)-benzaldehyde and 4-(thiophen-2-yl)-benzaldehyde;

(12) compounds of formula XXIV

wherein R₁₃ and R₁₄ are amino-protecting groups, which are differentfrom one another, selected from those mentioned under Process a),especially tert-lower alkoxycabonyl, such as tert-butoxycarbonyl, or anacylamino-protecting group, especially trifluoroacetyl; preferably R₁₃is trifluoroacetyl and R₁₄ is tert-butoxycarbonyl; (those compounds arecompounds of formula IX that are protected at both amino groups);

(13) compounds of formula XXV,

wherein R₁₄ is an amino-protecting group, as defined for compounds offormula XXIV, especially tert-butoxycarbonyl;

(14) compounds of formula XXVI,

wherein R₁₅ is an amino-protecting grdtip, especiallytert-butoxycarbonyl, and the remaining radicals are as defined forcompounds of formula I;

(15)1-[4-(2-tert-butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-N-(tert-butyloxycarbonyl)amino-2-N-[N-methoxycarbonyl-(L)-tert-leucyl]amino-6-phenyl-2-azahexane(as intermediate, but also pharmaceutically active).

When salt-forming groups are present, the compounds mentioned aboveunder (1) to (15) as starting materials may also be in the form of asalt.

EXAMPLES

The following Examples serve to illustrate the invention withoutlimiting the scope thereof:

Temperatures are indicated in degrees Celsius (° C.). Where notemperature is indicated, the reactions that follow are carried out atroom temperature. The R_(f) values, which indicate the ratio of theseepage propagation of the substance in question to the seepagepropagation of the eluant front, are determined on silica gel thin-layerplates (Merck, Darmstadt, Germany) by thin-layer chromatography (TLC)using the solvent systems mentioned in each case.

HPLC gradients used:

HPLC₂₀₋₁₀₀ 20% → 100% a) in b) for 20 min. HPLC_(20-100(12′)) 20% → 100%a) in b) for 12 min., then 8 min 100% a) HPLC₅₋₆₀  5% → 60% a) in b) for15 min.

Eluant a): acetonitrile+0.05% TFA; eluant b): water+0.05% TFA. Column(250×4.6 mm) packed with “reversed-phase” material C18-Nucleosil (5 μmmean particle size, silica gel covalently derivatised withoctadecylsilanes, Macherey & Nagel, Duren, Germany). Detection byUV-absorption at 254 nm. Retention times (t_(Ret)) are given in minutes.Flow rate 1 ml/min.

The other abbreviations used have the following meanings:

abs. absolute (indicates that solvent is anhydrous) anal. elementalanalysis Boc tert-butoxycarbonyl calc. calculated DBU1,8-diazabicyclo[5.4.0]undec-7-ene-(1,5-5) TLC thin-layer chromatographyDIPE diisopropyl ether DMF dimethylformamide DPPP[1,3-bis(diphenylphosphino)propane]nickel(II) chloride (Aldrich,Milwaukee, USA) EDC N-ethyl-N′-(3-dimethylaminopropyl)- carbodiimidehydrochloride ether diethyl ether FAB-MS fast atom bombardment massspectroscopy sat. saturated HOAc acetic acid HOBT1-hydroxy-benzotriazole HPLC High Performance Liquid ChromatographyHünig base N-ethyldiisopropylamine MeOH methanol min minute(s) NMMN-methylmorpholine Pd/C palladium on charcoal Pd(PPh₃)₄tetrakis(triphenylphosphine)palladium iso-PrOH isopropanol R_(f) ratioof seepage propagation to the eluant front in TLC SiO₂ silica gel m.p.melting point brine saturated sodium chloride solution TEA triethylamineTFA trifluoroacetic acid THF tetrahydrofuran (dist. oversodium/benzophenone) TPTU O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate p-TSA p-toluenesulfonic acid

Source of some amino acid derivatives used as starting materials:

(2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane J. Org. Chem. 50, 4615(1985)

(2R)-[(1′S)-(trifluoroacetyl)amino-2′-phenylethy]oxirane (EuropeanPatent Application EP 0 521 827, page 78, Ex. 16d))

N-methoxycarbonyl-(L)-valine (Preparation see Chem. Lett. 705 (1980))

N-ethoxycarbonyl-(L)-valine (Preparation see J. Org. Chem. 60, 7256(1995))

N-methoxycarbonyl-(L)-iso-leucine (Preparation see Chem. Lett. 705(1980))

Example 11-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-valyl)amino]-6-phenyl-2-azahexane

With the exclusion of moisture, 735 mg (4.20 mmol) ofN-methoxycarbonyl-(L)-valine (see EP 0 604 368, Example 2b)), 1548 mg(8.07 mmol) of EDC and 654 mg (4.844 mmol) of HOBT areplaced in 10 ml ofDMF. 1.13 ml (8.07 mmol) of TEA are added to the white suspension andthe mixture is stirred at room temperature for 30 min. Then 595 mg (1.62mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanedissolved in 1 ml of DMF are added and the mixture is stirred ovenightto complete the reaction. The reaction mixture is concentrated byevaporation; the resulting oil is dissolved in methylene chloride andwashed with 10% citric acid solution, sat. NaHCO₃ solution and brine.The aqueous phases are extracted 2× with methylene chloride; thecombined organic phases are filtered through cotton wadding andconcentrated by evaporation. Column chromatography (SiO₂;CH₂Cl₂/MeOH/H₂O/HOAc 85:13:1.5:0.5) and precipitation with DIPE from aconcentrated solution in methylene chloride yield the title compound:TLC: R_(f)=0.57 (CH₂Cl₂/MeOH/H₂O/HOAc 85:13:1.5:0.5); HPLC₂₀₋₁₀₀:t_(Ret)=13.0; FAB MS (M+H)⁺=683.

The starting material is prepared as follows:

1a) 4-(Thiazol-5-yl)-benzaldehyde

In a bomb tube, a mixture of 3.7 g (20 mmol) of 4-bromobenzaldehyde(Fluka, Buchs, Switzerland), 6.64 ml (93 mmol) of thiazole, 2.94 g ofpotassium acetate and 1.16 g (1 mmol) of Pd(PPh₃)₄ in 50 ml ofdimethylacetamide is stirred at 150° C. for 12 hours. The reactionmixture is concentrated by evaporation. Water is added to the residueand the mixture is extracted 3× with methylene chloride. The organicphases are filtered through cotton wadding, concentrated by evaporationand chromatographed (SiO₂; hexane/ethyl acetate 1:2), yielding the titlecompound: HPLC₂₀₋₁₀₀: t_(Ret)=11.4; ¹H-NMR (CD₃OD) δ 9.98 (s, HCO), 9.03(s, H(2)^(thiazole)), 8.32 (s, H(4)^(thiazole)), 7.95 and 7.85 (2d, J=8,each 2H); additionally also signals of the hydrate (≈12%): 8.92 (s,H(2)^(thiazole)), 8.15 (s, H(4)^(thiazole)), 7.62 and 7.53 (2d, J=8,each 2H), 5.54 (s, HC(OH)₂).

1b)N-1-(tert-Butoxycarbonyl)-N-2-{[4-(thiazol-5-yl)-phenyl]-methylidene}-hydrazone

A solution of 1.22 g (6.45 mmol) of 4-(thiazol-5-yl)-benzaldehyde and1.12 g (6.14 mmol) of tert-butyl carbazate (Fluka, Buchs, Switzerland)in 40 ml of ethanol is stirred at 80° C. for 12 hours. Cooling andcrystallisation by the addition of 60 ml of water at 0° C. yield thetitle compound: m.p: 170-171° C.; HPLC₂₀₋₁₀₀: t_(Ret)=13.5.

1c) N-1-(tert-Butoxycarbonyl)-N-2-[4-(thiazol-5-yl)-benzyl]-hydrazine

Under a nitrogen atmosphere, 20.4 g (67.2 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-{[4-(thiazol-5-yl)-phenyl]-methylidene}-hydrazoneare placed in 120 ml of THF, and 4.67 g (70.7 mmol; 95%) of sodiumcyanoborohydride are added. A solution of 12.8 g (67.2 mmol) ofp-toluenesulfonic acid monohydrate in 120 ml of THF (pH 3-4) is thenadded dropwise thereto. After 7 hours, water and ethyl acetate are addedand the aqueous phase is separated off and extracted a further 2× withethyl acetate. The organic phases are washed with brine, sat. NaHCO₃solution and brine, dried (Na₂SO₄) and concentrated by evaporation. Tothe resulting viscous oil there are added 80 ml of dichloroethane and 80ml of 1N NaOH solution (foams) and the mixture is boiled under refluxfor 7 hours. The reaction mixture is cooled and diluted with methylenechloride and water; the aqueous phase is separated off and extracted 2×with methylene chloride. The organic phases are dried (Na₂SO₄),concentrated by evaoporation and chromatographed (SiO2; hexane/ethylacetate 2:1). Stirring in hexane yields the title compound: m.p: 93-95°C.; TLC: R_(f)=0.12 (hexane/ethyl acetate 2:1); Anal. (C₁₅H₁₉N₃O₂S)calc. C, 58.99, H, 6.27, N, 13.76, S, 10.50; found C, 58.98, H, 6.34, N,13.64, S, 10.66; HPLC₂₀₋₁₀₀: t_(Ret)=10.1.

1d)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexane

A suspension of 1.21 g (4.6 mmol) of(2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane and 1.4 g (4.6 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(thiazol-5-yl)-benzyl]-hydrazine in 25ml of iso-PrOH is heated at boiling overnight. The reaction mixture iscooled and water is added. The supernatant phase is decanted off fromthe oil that has separated out; the oil is dried in vacuo andchromatographed (SiO₂; methylene chloride/methanol 30:1), yielding thetitle compound: TLC: R_(f)=0.2 (methylene chloride/methanol 30:1);HPLC₂₀₋₁₀₀: t_(Ret)=17.2.

1e)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexane

A solution of 1.14 g (2.0 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexanein 100 ml of formic acid is stirred at room temperature for 3 hours andthen concentrated by evaporation. Sat. NaHCO₃ solution and methylenechloride are added to the residue; the aqueous phase is separated offand extracted 2× with methylene chloride. The organic phases are treatedwith brine, filtered through cotton wadding and concentrated byevaporation to form the title compound which is used further directly.

Example 21-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-valyl)-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino6-phenyl-2-azahexane

Under an argon atmosphere, 344 mg of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexaneand 191 μl (1.74 mmol) of NMM in 5.6 ml of DMF are added to 122 mg(0.696 mmol) of N-methoxy-carbonyl-(L)-valine and 173 mg (0.58 mmol) ofTPTU in 2.9 ml of DMF and the mixture is stirred at room temperature for16 hours. The reaction mixture is poured into ice-water, stirred for 30min and filtered. Column chromatography of the residue (SiO₂; methylenechloride/THF 4:1) and stirring in ether yield the title compound: m.p:134-135° C.; HPLC₂₀₋₁₀₀: t_(Ret)=14.0; FAB MS (M+H)⁺=697.

The starting material is prepared as follows:

2a)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-(tri-fluoroacetyl)amino-6-phenyl-2-azahexane

A suspension of 5.32 g (20.5 mmol) of(2R)-[(1′S)-(trifluoroacetyl)amino-2′-phenylethyl]-oxirane and 5.7 g(18.6 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(thiazol-5-yl)-benzyl]-hydrazine(Example 1c) in 95 ml of iso-PrOH is heated at boiling for 8 hours.After cooling, the reaction mixture is partially concentrated byevaporation and left to stand at 0° C., resulting in the crystallisationof the title compound which is filtered off with suction and dried. TLC:R_(f)=0.39 (methylene chloride/THF 10:1); HPLC₂₀₋₁₀₀: t_(Ret)=16.5; FABMS (M+H)⁺=565. Further product can be obtained from the mother liquor byboiling again with(2R)-[(1′S)-(trifluoroacetyl)amino-2′-phenylethyl]oxirane in iso-PrOHand column chromatography (SiO₂; methylene chloride/THF 15:1).

2b)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane

100 ml of a 1N K₂CO₃ solution are added dropwise to a solution of 5.646g (10.0 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-(trifluoroacetyl)-amino-6-phenyl-2-azahexanein 100 ml of methanol and the mixture is stirred at 70° C. for 15 hours.Methylene chloride and water are added; the aqueous phase is separatedoff and extracted 2× with methylene chloride. The organic phases arewashed 2× with water, dried (Na₂SO₄) and concentrated by evaporation,yielding the title compound: Anal. (C₂₅H₃₂N₄O₃S (0.53 H₂O)) calc. C,62.80, H, 6.97, N, 11.72, S, 6.71, H₂O 2.00: found C, 63.2, H, 7.01, N,11.57, S, 6.49, H₂O 1.98; HPLC₂₀₋₁₀₀: t_(Ret)=11.5.

2c)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

Under an nitrogen atmosphere, 1.36 g (7.2 mmol) ofN-methoxycarbonyl-(L)-tert-leucine (Example 2e), 2.59 g (13.5 mmol) ofEDC and 1.22 g (9.0 mmol) of HOBT are dissolved in 20 ml of DMF. After15 min, 3.79 ml (27 mmol) of TEA are added and then a solution of 2.11 g(4.5 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexanein 41 ml of DMF is added dropwise. After 3 hours the reaction mixture isconcentrated by evaporation. The resulting oil is dissolved in ethylacetate and a small amount of THF and washed with 2×water, sat. NaHCO₃solution, 2×water and brine. The aqueous phases are extracted with ethylacetate; the combined organic phases are dried (Na₂SO₄) and concentratedby evaporation. Column chromatography (SiO₂; methylene chloride/THF 5:1)and crystallisation from ethyl acetate/DIPE yield the title compound:HPLC₂₀₋₁₀₀: t_(Ret)=16.0; FAB MS (M+H)⁺=640.

2d)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

742 mg (1.16 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexaneand 12 ml of formic acid are stirred at room temperature for 7 hours andthen concentrated by evaporation. Sat. NaHCO₃ solution and ethyl acetateare added to the residue; the aqueous phase is separated off andextracted with ethyl acetate. The organic phases are treated with waterand brine, dried (Na₂SO₄) and concentrated by evaporation, yielding thetitle compound which is used further directly.

2e) N-(Methoxycarbonyl)-(L)-tert-leucine

23.5 ml (305 mmol) of methyl chloroformate are added over a period of 20min to a solution of 20 g (152 mmol) of (L)-tert-leucine(=2(S)-amino-3,3-dimethyl-butyric acid=(L)-α-tert-butylglycine; Fluka,Buchs/Switzerland) in a mixture of 252 ml (504 mmol) of 2N aqueoussodium hydroxide solution and 80 ml of dioxane and the reaction solutionis heated at 60° C. for 14 hours. After cooling to room temperature, thereaction solution is washed 2× with methylene chloride. The aqueousphase is acidified to pH 2 with 4N aqueous hydrochloric acid andextracted three times with ethyl acetate. The organic extracts arecombined, dried (Na₂SO₄) and concentrated by evaporation, the productbeginning to solidify. Digestion of the solidified solid with hexaneyields the title compound in the form of a white powder. M.p. 106-108°C.

Example 31-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane

Under an argon atmosphere, 292 mg of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leuyl)amino-6-phenyl-2-azahexane(Example 2d) and 165 μl (1.5 mmol) of NMM in 4.8 ml of DMF are added to113.5 mg of N-methoxycarbonyl-(L)-tert-leucine (Example 2e) and 149 mg(0.50 mmol) of TPTU in 2.5 ml of DMF and the mixture is stirred at roomtemperature for 14 hours. The reaction mixture is poured into 0.2 literof ice-water, stirred for 45 min and filtered. Column chromatography ofthe residue (SiO₂; methylene chloride/ethanol 20:1) and crystallisationfrom ethyl acetate/ether/hexane yield the title compound: m.p: 207-209°C.; TLC: R_(f)=0.25 (methylene chloride/ethanol 20:1); HPLC₂₀₋₁₀₀:t_(Ret)=14.7; FAB MS (M+H)⁺=711.

Example 41-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 292 mg of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane(Example 2d) and 165 μl (1.5 mmol) of NMM in 4.8 ml of DMF are added to113 mg of N-methoxycarbonyl-(L)-iso-leucine and 149 mg (0.50 mmol) ofTPTU in 2.5 ml of DMF, and the midure is stirred at room temperature for14 hours and worked up analogously to Example 3, yielding the titlecompound: m.p: 139-141° C.; TLC: R_(f)=0.7 (methylene chloride/methanol10:1); HPLC₂₀₋₁₀₀: t_(Ret)=14.6; FAB MS (M+H)⁺=711.

Example 51-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-S-methylcysteinyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 292 mg of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane(Example 2d) and 165 μl (1.5 mmol) of NMM in 4.8 ml of DMF are added to116 mg (0.60 mmol) of N-methoxycarbonyl-(L)-S-methylcysteine and 149 mg(0.50 mmol) of TPTU in 2.5 ml of DMF, and the mixture is stirred at roomtemperature for 5 hours and worked up analogously to Example 3, yieldingthe title compound: TLC: R_(f)=0.4 (methylene chloride/methanol 10:1);HPLC₂₀₋₁₀₀: t_(Ret)=13.6; FAB MS (M+H)⁺=715.

The starting material is prepared as follows:

5a) N-methoxycarbonyl-(L)-S-methylcysteine

With ice-cooling, 16.8 g (177.5 mmol) of chloroformic acid methyl esterare added dropwise to a solution of 12.0 g (88.8 mmol) ofS-methyl-(L)-cysteine ((S)-2-amino-3-methylmercapto-propionic acid:Fluka; Buchs/Switzeriand) in 150 ml of 2N sodium hydroxide solution and18 ml of dioxane and the mixture is stirred at 70° C. overnight tocomplete the reaction. The reaction mixture is diluted with 150 ml ofmethylene chloride; the aqueous phase is separated off, acidified with1N HCl and extracted 3× with ethyl acetate. Drying (Na₂SO₄) andconcentration of the ethyl acetate phases by evaporation yield the titlecompound: FAB MS (M+H)⁺=194.

Example 61-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-ethoxycarbonyl-(L)-valyl)-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 344 mg of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane(Example 2d) and 191 μl (1.74 mmol) of NMM in 5.6 ml of DMF are added to132 mg (0.7 mmol) of N-ethoxycarbonyl-(L)-valine (EP 0 604 368, Example9a) and 173 mg (0.58 mmol) of TPTU in 2.9 ml of DMF, and the mixture isstirred at room temperature overnight and worked up analogously toExample 3, yielding the title compound: TLC: R_(f)=0.45 (methylenechloride/THF 4:1); HPLC₂₀₋₁₀₀: t_(Ret)=14.7; FAB MS (M+H)⁺=711.

Example 71-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxyarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Under argon, 213 mg (1.13 mmol) of N-methoxycarbonyl-(L)-tert-leucine(Example 2e), 431 mg (2.25 mmol) of EDC and 304 mg (2.25 mmol) of HOBTare placed in 18 ml of DMF. After 15 min, 627 μl (4.5 mmol) of TEA and0.75 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneare added. After 2 hours, water and ethyl acetate are added; the aqueousphase is separated off and extracted a further 2× with ethyl acetate.The organic phases are washed 2× with water, sat. NaHCO₃ solution2×water and brine, dried (Na₂SO₄) and concentrated by evaporation.Column chromatography (SiO₂; methylene chloride/THF 5:1) andcrystallisation from ether yield the title compound: m.p: 200-201 ° C.;HPLC₂₀₋₁₀₀: t_(Ret)=14.0; FAB MS (M+H)⁺=697.

The starting material is prepared as follows:

7a)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxecarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 2.66 g (15.2 mmol) ofN-methoxycarbonyl-(L)-valine, 5.46 g (28.5 mmol) of EDC and 2.57 g (19mmol) of HOBT are dissolved in 42 ml of DMF. 7.9 ml (57 mmol) of TEA areadded and after 20 min a solution of 4.46 g (9.5 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane(Example 2b) in 85 ml of DMF is added dropwise. After 1.5 hours, thereaction mixture is worked up analogously to Example 2c. Crystallisationfrom THF/ether yields the title compound: m.p: 114-115° C.; HPLC₂₀₋₁₀₀:t_(Ret)=15.1; FAB MS (M+H)⁺=626.

7b)1-[4-(Thiazol-5-yl)-phenyl-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

1.25 g (2.0 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneand 18 ml of formic acid are reacted analogously to Example 2d to formthe title compound: HPLC₂₀₋₁₀₀: t_(Ret)=10.0.

Example 81-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-ethoxycarbonyl-(L)-valyl)-amino-5(S)-N-(N-metboxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Analogously to Example 7, 213 mg (1.13 mmol) ofN-ethoxycarbonyl-(L)-valine, 431 mg (2.25 mmol) of EDC and 304 mg (2.25mmol) of HOBT in 18 ml of DMF and 627 μl (4.5 mmol) of TEA are reactedwith 0.75 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane(Example 7b) to form the title compound: m.p: 243-244° C.; HPLC₂₀₋₁₀₀:t_(Ret)=14.0; FAB MS (M+H)⁺=697.

Example 91-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 0.6 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneand 198 μl (1.8 mmol) of NMM in 5.8 ml of DMF are added to 136 mg (0.72nmol) of N-methoxycarbonyl-(L)-iso-leucine and 179 mg (0.60 mmol) ofTPTU in 3 ml of DMF and the mixture is stirred at room temperature for14 hours and worked up analogously to Example 3, yielding the titlecompound: TLC: R_(f)=0.59 (methylene chloride/THF 3:1); HPLC₂₀₋₁₀₀:t_(Ret)=14.0; FAB MS (M+H)⁺=697.

Example 101-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-S-methylcysteinyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 0.58 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneand 191 μl (1.74 mmol) of NMM in 5.6 ml of DMF are added to 134 mg(0.696 mmol) of N-methoxycarbonyl-(L)-S-methylcysteine (Example 5a) and173 mg (0.58 mmol) of TPTU in 2.9 ml of DMF and the mixture is stirredat room temperature for 15 hours and worked up analogously to Example 3,yielding the title compound: TLC: R_(f)=0.17 (methylene chloride/THF4:1); HPLC₂₀₋₁₀₀: t_(Ret)=13.0; FAB MS (M+H)⁺=701.

Example 111-[4-(Thiazol-5-yl)-phenyl]4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under argon, 0.5 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexaneand 165 μl (1.5 mmol) of NMM in 4.8 ml of DMF are added to 113.5 mg(0.60 mmol) of N-methoxycarbonyl-(L)-tert-leucine (Example 2e) and 149mg (0.50 mmol) of TPTU in 2.5 ml of DMF and the mixture is stirred atroom temperature for 14 hours. Ice-water and and ethyl acetate areadded; the aqueous phase is separated off and extracted with ethylacetate. The organic phases are washed 2× with water and brine, dried(Na₂SO₄) and concentrated by evaporation. Column chromatography (SiO₂;ethyl acetate) and crystallisation from ethyl acetate/ether/hexane yieldthe title compound: TLC: R_(f)=0.42 (methylene chloride/ethanol 10:1);HPLC₂₀₋₁₀₀: t_(Ret)=14.8; FAB MS (M+H)⁺=711.

The starting material is prepared as follows:

11a)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 1.36 g (7.2 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 2.59 g (13.5 mmol) of EDC and 1.22 g(9 mmol) of HOBT are dissolved in 20 ml of DMF. After 30 min, 3.79 ml(27 mmol) of TEA are added and a solution of 2.11 g (4.5 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane(Example 2b) in 40 ml of DMF are added dropwise. After 3 hours, thereaction mixture is worked up analogously to Example 2c to form thetitle compound: m.p: 163-166° C.; Anal. (C₃₃H₄₅N₅O₆S (0.14 H₂O)) calc.C, 61.71, H, 7.11, N, 10.90, S, 4.99, H₂O 0.39: found C, 61.61, H, 7.10,N, 10.79, S, 4.76, H₂O 0.4; HPLC₂₀₋₁₀₀: t_(Ret)=16.0; FAB MS (M+H)⁺=640.

11b)1-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydrox-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

320 mg (0.50 mmol) of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexaneand 6 ml of formic acid are reacted analogously to Example 2d to formthe title compound which is used further directly.

Example 121-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Analogously to Example 7, 140 mg (0.80 mmol) ofN-methoxycarbonyl-(L)-valine, 288 mg (1.5 mmol) of EDC and 135 mg (1.0mmol) of HOBT in 2 ml of DMF and 418 μL of TEA are reacted with 0.5 mmolof1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanein 5 ml of DMF to form the title compound: m.p: 202-204° C.; HPLC₂₀₋₁₀₀:t_(Ret)=14.0; FAB MS (M+H)⁺=697.

Example 131-[4-(Thiazol-5-yl)-phenyl-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxy-carbonyl-(L)-iso-leucyl)amino]-6-phenyl-2-azahexane

Analogously to Example 7, 175 mg (0.92 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 332 mg (1.7 mmol) of EDC and 156 mg(1.15 mmol) of HOBT in 2.5 ml of DMF and 483 μl (3.47 mmol) of TEA arereacted with 0.578 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane(Example 11 b) in 5.2 ml of DMF to form the title compound: m.p:213-216° C.; HPLC₂₀₋₁₀₀: t_(Ret)=14.7; FAB MS (M+H)⁺=711.

Example 141-[4-(Thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-ethoxycarbonyl-(L)-valyl)-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Analogously to Example 7, 175 mg (0.92 mmol) ofN-ethoxycarbonyl-(L)-valine, 332 mg (1.7 mmol) of EDC and 156 mg (1.15mmol) of HOBT in 2.5 ml of DMF and 483 μl (3.47 mmol) of TEA are reactedwith 0.578 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane(Example 11b) in 5.2 ml of DMF to form the title compound: m.p: 200-203°C.; HPLC₂₀₋₁₀₀: t_(Ret)=14.6; FAB MS (M+H)⁺=711.

Example 151-[4-(Thiazol-5-yl)-phenyl]4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-S-methylcysteinyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 0.5 mmol of1-[4-(thiazol-5-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane(Example 11b) and 165 μl (1.5 mmol) of NMM in 4.8 ml of DMF are addedwith ice cooling to 116 mg (0.60 mmol) ofN-methoxycarbonyl-(L)-S-methylcysteine (Example 5a) and 149 mg (0.50mmol) of TPTU in 2.5 ml of DMF and the mixture is stirred at roomtemperature for 12 hours. Water and ethyl acetate are added; the aqueousphase is separated off and extracted a further 2× with ethyl acetate.The organic phases are washed 2× with water and brine, dried (Na₂SO₄)and partially concentrated by evaporation. The addition of ether causesthe titie compound to crystallise: m.p: 179-181° C.; TLC: R_(f)=0.67(methylene chloride/ethanol 10:1); HPLC₂₀₋₁₀₀: t_(Ret)=13.6; FAB MS(M+H)⁺=715.

Example 161-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane

Under an argon atmosphere, 2.58 g (13.7 mmol) ofN-methoxycarbonyl-(L)-tert-leucine and 4.09 g (13.7 mmol) of TPTU aredissolved in 15.5 ml of DMF; 5.7 ml (24.8 mmol) of Hünig base are addedwith cooling and the mixture is stirred for 10 min. Then a solution of2.29 g (6.20 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanein 15.5 ml of DMF is added and the mixture is stirred at roomtemperature for 16 hours. The light-yellow reaction solution is pouredinto ice-water; ethyl acetate is added and the mixture is stirred for 30min. The aqueous phase is separated off and extracted a further 2× withethyl acetate. The organic phases are extracted 2× with water, sat.NaHCO₃ solution and 2× with brine, dried (Na₂SO₄) and concentrated byevaporation. Column chromatography (SiO₂; hexane/ethyl acetate 1:3) andcrystallisation from methylene chloride/DIPE yield the title compound:TLC: R_(f)=0.18 (hexane/ethyl acetate 1:3); HPLC_(20-100(12′)):t_(Ret)=11.0; FAB MS (M+H)⁺=711; [α]⁰.(c=0.6, ethanol)=−46°.

The starting material is prepared as follows:

16a) 4-(Thiazol-2-yl)-benzaldehyde

Under argon, 9.2 g (379 mmol) of magnesium are placed in 84 ml of THFand heated to 60° C. A solution of 82.6 g (357 mmol) of4-bromobenzaldehyde dimethyl acetal (for preparation see J. Org. Chem.56, 4280 (1991)) in 677 ml of THF is added dropwise thereto within aperiod of 30 min and the mixture is stirred at boiling temperature for afurther 40 min. The Grignard solution is cooled, decanted into adropping funnel and added dropwise within a period of 30 min to areddish suspension of 31.7 ml (338 mmol) of 2-bromothiazole (Fluka,Buchs, Switzerland) and 5.39 g (9.95 mmol) of DPPP in 1.68 liters ofTHF. The mixture is stirred at room temperature for 12 hours; a further5.39 g of DPPP are added and the mixture is stirred for a further 7hours. 840 ml of water are added and the mixture is stirred for 10 min;the THF is evaporated off using a rotary evaporator and the residue isstirred for 1.5 hours in 1.0 liter of ether and 340 ml of 2N HCl. Theaqueous phase is separated off and extracted 2× with ethyl acetate. Theorganic phases are washed 2× with 0.5N HCl, water, sat. NaHCO₃ solution,water and brine, dried (Na₂SO₄) and concentrated by evaporation.Chromatography (SiO₂; hexane/ethyl acetate 4:1) and digestion in hexaneyield the title compound: TLC: R_(f)=0.21 (hexane/ethyl acetate 3:1);m.p: 91-92° C.; Anal. (C₁₀H₇NOS) calc. C, 63.47, H, 3.73, N, 7.40, S,16.94: found C, 63.14, H, 3.79, N, 7.27, S, 17.08; ¹H-NMR (CDCl₃) δ10.05 (s, HCO), 8.15 (d, J=8, 2H), 7.95 (m, 3H), 7.45 (d, J=3,1H).

16b)N-1-(tert-Butoxycarbonyl)-N-2-{[4-(thiazol-2-yl)-phenyl]-methylidene}-hydrazone

A solution of 27.6 g (145 mmol) of 4-(thiazol-2-yl)-benzaldehyde and19.7 g (149 mmol) of tert-butyl carbazate in 920 ml of ethanol isstirred at 80° C. for 18 hours. Cooling, concentration by evaporationand stirring from DIPE yield the title compound: TLC: R_(f)=0.31(toluene/ethyl acetate 3:1); HPLC₂₀₋₁₀₀: t_(Ret)=14.5.

16c) N-1-(tert-Butoxycarbonyl)-N-2-[4-(thiazol-2-yl)-benzyl]-hydrazine

Under a nitrogen atmosphere, 77.6 g (256 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-{[4-(thiazol-2-yl)-phenyl]-methylidene}-hydrazoneare placed in 450 ml THF, and 16.9 g (257 mmol; 95%) of sodiumcyanoborohydride are added. A solution of 49.6 g (261 mmol) ofp-toluenesulfonic acid monohydrate in 450 ml of THF (pH 3-4) is addeddropwise thereto. After 17 hours, a further 3.38 g of sodiumcyanoborohydride are added; the mixture is adjusted to pH 3-4 withp-toluenesulfonic acid monohydrate solution and stirred for 3 hours tocomplete the reaction. Water and ethyl acetate are added; the aqueousphase is separated off and extracted a further 2× with ethyl acetate.The organic phases are washed with brine, sat. NaHCO₃ solution and2×brine, dried (Na₂SO₄) and concentrated by evaporation. The resultingviscous oil is taken up with 300 ml of 1,2-dichloroethane; 300 ml of 1NNaOH solution are slowly added (foams) and the mixture is boiled underreflux for 3.5 hours. The mixture is cooled and diluted with methylenechloride and water; the aqueous phase is separated off and extracted 2×with methylene chloride. The organic phases are dried (Na₂SO₄),concentrated by evaporation and chromatographed (SiO₂; toluene/acetone9:1→6:1). Stirring in hexane yields the title compound: TLC: R_(f)=0.3(hexane/ethyl acetate 3:2); HPLC₂₀₋₁₀₀: t_(Ret)=11.1.

16d)1-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexane

A solution of 6.00 g (22.8 mmol) of(2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane and 5.37 g (17.6 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(thiazol-2-yl)-benzyl]-hydrazine in 550ml of iso-PrOH is heated at boiling overnight. The reaction mixture iscooled to room temperature, poured into 0.2 liter of water, withstirring, and cooled with ice. Filtration with suction, washing withwater and ether and drying yield the title compound: TLC: R_(f)=0.36(hexanel/acetone 3:2); HPLC_(20-100(12′)): t_(Ret)=12.7. Further productcan be isolated from the mother liquor by chromatography (SiO₂;hexane/acetone 3:2).

16e)1-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexane

A solution of 4.3 g (7.56 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexanein 378 ml of formic acid is stirred at room temperature for 3.5 hours(argon) andqhen concentrated by evaporation. Sat. NaHCO₃ solution andmethylene chloride are added to the residue; the aqueous phase isseparated off and extracted 2× with methylene chloride. The organicphases are treated with brine, dried (Na₂SO₄) and concentrated byevaporation to form the title compound: HPLC_(20-100(12′)): t_(Ret)=6.8.

Example 171-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxylcarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 294 mg of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneand 165 μl (1.5 mmol) of NMM in 4.8 ml of DMF are added to 113.5 mg(0.60 mmol) of N-methoxycarbonyl-(L)-tert-leucine (Example 2e) and 149mg (0.50 mmol) of TPTU in 2.5 ml of DMFat 0° C. and the mixture isstirred at room temperature for 18 hours. Water and ethyl acetate areadded; the aqueous phase is separated off and extracted a further 2×with ethyl acetate. The organic phases are washed 2× with water, sat.NaHCO₃ solution, water and brine, dried (Na₂SO₄) and concentrated byevaporation. Column chromatography (SiO₂; methylene chloride/THF 4:1)and precipitation with hexane from a concentrated solution in methylenechloride yield the title compound: HPLC₂₀₋₁₀₀: t_(Ret)=14.5; FAB MS(M+H)⁺=697.

The starting material is prepared as follows:

17a)1-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-(trifluoroacetyl)amino-6-phenyl-2-azahexane

With the exclusion of air, 4.8 g (18.5 mmol) of(2R)-[(1′S)-(trifluoroacetyl)amino-2′-phenyl-ethyl]oxirane and 3.78 g(12.4 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(thiazol-2-yl)-benzyl]-hydrazine(Example 16c) in 62 ml of iso-PrOH are heated at boiling for 10 hours.Cooling the reaction mixture, filtration and washing with ether yieldthe title compound: Anal. (C₂₇H₃₁N₄F₃O₄S) calc. C, 57.44, H, 5.53, N,9.92, F, 10.09, S, 5.68: found C, 57.27, H, 5.49, N, 9.91, F, 9.94, S,5.70; HPLC₂₀₋₁00: t_(Ref)=16.9; FAB MS (M+H)⁺=565. Further product canbe isolated from the filtrate by concentration by evaporation, columnchromatography (SiO₂; methylene chloride/THF 25:1) and stirring fromether/ethyl acetate.

17b)1-[4-(Thiazol-2-yl)phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane

55 ml of a 1N K₂CO₃ solution are added dropwise to 3.12 g (5.5 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-(trifluoroacetyl)amino-6-phenyl-2-azahexanein 55 ml of methanol and the mixture is stirred at 70° C. for 9 hours.The mixture is cooled and ≈30 ml of methanol are evaporated off;methylene chloride and water are added and the aqueous phase isseparated off and extracted with methylene chloride; the organic phasesare washed with water, dried (Na₂SO₄) and concentrated by evaporation,yielding the title compound: HPLC₂₀₋₁₀₀: t_(Ret)=1.9; FAB MS (M+H)⁺=469.

17c)1-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 1.4 9 (8.0 mmol) ofN-methoxycarbonyl-(L)-valine, 2.87 g (15 mmol) of EDC and 1.35 g (10mmol) of HOBT are dissolved in 22 ml of DMF. After 45 min, 4.2 ml (30mmol) of TEA are added and then a solution of 2.34 g (5.0 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxyarbonyl)amino-5(S)-amino-6-phenyl-2-azahexanein 45 ml of DMF is added dropwise. After 1.5 hours, the reaction mixtureis concentrated by evaporation; the residue is taken up in methylenechloride and washed with water, sat. NaHCO₃ solution, water and brine.The aqueous phases are extracted 2× with methylene chloride; thecombined organic phases are dried (Na₂SO₄) and concentrated byevaporation. Column chromatography (SiO₂; methylene chloride/ethylacetate 2:1) and crystallisation from ethyl acetate/ether yield thetitle compound: m.p: 178-179° C.; HPLC₂₀₋₁₀₀: t_(Ret)=15.8.

17d)1-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

0.94 g (1.5 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneand 18 ml of formic acid are stirred at room temperature for 6 hours andworked up analogously to Example 2d to form the title compound: FAB MS(M+H)⁺=526.

Example 181-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-vaylyl)amino-6-phenyl-2-azahexane

Analogously to Example 7, 106 mg (0.56 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 201 mg (1.05 mmol) of EDC and 95 mg(0.7 mmol) of HOBT in 4.6 ml of DMF and 293 μl (2.1 mmol) of TEA arereacted with 0.35 mmol of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneto form the title compound: m.p: 227-229° C.; HPLC₂₀₋₁₀₀: t_(Ret)=14.5;FAB MS (M+H)⁺=697.

Example 19 1-[4-(Thiazol-2-yl)-phenyl]4(S)-hydroxy-2-N-(N-ethoxycarbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Analogously to Example 7,106 mg (0.56 mmol) ofN-ethoxycarbonyl-(L)-valine, 201 mg (1.05 mmol) of EDC and 95 mg (0.7mmol) of HOBT in 4.6 ml of DMF and 293 μl (2.1 mmol) of TEA are reactedwith 0.35 mmol of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneto form the title compound: Anal. (C₃₅H₄₈N₆O₇S (0.20 H₂O)) calc. C,60.01, H, 6.96, N, 12.00, S, 4.58, H₂O 0.51: found C, 60.07, H, 6.78, N,11.93, S, 4.70, H₂O 0.52; HPLC20-100: t_(Ret)=14.6; FAB MS (M+H)⁺=697.

Example 201-[4-(Thiazol-2-yl)-phenyl-4(S-hydroxy-5(S)-2,5-bis[N-(N-methoxy-carbonyl-(L)-valyl)amino]-6-phenyl-2-azahexane

Analogously to Example 7, 140 mg (0.80 mmol) ofN-methoxycarbonyl-(L)-valine, 288 mg (1.5 mmol) of EDC and 135 mg (1.0mmol) of HOBT in 2.2 ml of DMF and 418 μl (3.0 mmol) of TEA are reactedwith 0.5 mmol of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanein 4.5 ml of DMF to form the title compound: m.p: 207-210° C.;HPLC₂₀₋₁₀₀: t_(Ret)=13.8; FAB MS (M+H)⁺=683.

Example 211-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 294 mg of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexaneand 165 μl (1.5 mmol) of NMM in 4.8 ml of DMF are added to 113.5 mg(0.60 mmol) of N-methoxycarbonyl-(L)-tert-leucine (Example 2e) and 149mg (0.50 mmol) of TPTU in 2.5 ml of DMF at 0° C. and the mixture isstirred at room temperature for 16 hours. Ice-water and ethyl acetateare added; the aqueous phase is separated off and extracted with ethylacetate. The organic phases are washed 2× with water and brine, dried(Na₂SO₄) and concentrated by evaporation. Column chromatography (SiO₂;ethyl acetate) and crystallisation from ethyl acetate/ether/hexane yieldthe title cortfpound: Anal. (C₃₆H₅₀N₆O₇S (1.4% H₂O)) calc. C, 59.97, H,7.15, N, 11.66, S, 4.45: found C, 59.99, H, 7.18, N, 11.35, S, 4.59;TLC: R_(f)=0.51 (methylene chloride/THF 3:1); HPLC₂₀₋₁₀₀: t_(Ret)=15.2;FAB MS (M+H)⁺=711.

The starting material is prepared as follows:

21a)1-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 938 mg (4.96 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 1.78 g (9.3 mmol) of EDC and 838 mg(6.2 mmol) of HOBT are dissolved in 13.7 ml of DMF. After 30 min, 2.6 ml(18.6 mmol) of TEA are added and then a solution of 1.45 g (3.1 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane(Example 17b) in 28 ml of DMF is added dropwise thereto. After 3 hoursthe reaction mixture is concentrated by evaporation; the residue istaken up in ethyl acetate and a small amount of THF and washed withwater, sat. NaHCO₃ solution, water and brine. The aqueous phases areextracted with ethyl acetate; the combined organic phases are dried overNa₂SO₄ and concentrated by evaporation. Column chromatography (SiO₂;methylene chloride/THF 5:1) and stirring from ethyl acetate/DIPE yieldthe title compound: HPLC₂₀₋₁₀₀: t_(Ret)=16.3; FAB MS (M+H)⁺=640.

21b)1-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

761 mg (1.2 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexaneand 12 ml of formic acid are stirred at room temperature for 7 hours andworked up analogously to Example 2d to form the title compound.

Example 221-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-ethoxycarbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 321 mg (0.60 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane(Example 21b) and 182 mg (1.8 mmol) of NMM in 5.8 ml of DMF are added to136 mg (0.72 mmol) of N-ethoxycarbonyl-(L)-valine and 178 mg (0.60 mmol)of TPTU in 3 ml of DMF and the mixture is stirred at room temperaturefor 15 hours. The reaction mixture is poured into ice-water, stirred for30 min and filtered. Crystallisation from THF with DIPE and hexaneyields the title compound: m.p.: 209-211° C.; HPLC₂₀₋₁₀₀: t_(Ret)=15.2;FAB MS (M+H)⁺=711.

Example 231-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 321 mg (0.60 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane(Example 21b) and 182 mg (1.8 mmol) of NMM in 5.8 ml of DMF are added to126 mg (0.72 mmol) of N-methoxycarbonyl-(L)-valine and 178 mg (0.60mmol) of TPTU in 3 ml of DMF; the mixture is stirred at room temperaturefor 15 hours and worked up analogously to Example 3. TLC: R_(f)=0.15(methylene chloride/THF 4:1); HPLC₂₀₋₁₀₀: t_(Ret)=14.5; FAB MS(M+H)⁺=697.

Example 241-[4-(Thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-S-methylcysteinyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Under an argon atmosphere, 303 mg (0.50 mmol) of1-[4-(thiazol-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane(Example 21b) and 165 μl (1.5 mmol) of NMM in 5 ml of DMF are added to116 mg (0.60 mmol) of N-methoxycarbonyl-(L)-S-methylcysteine (Example5a) and 149 mg (0.50 mmol) of TPTU in 2.5 ml of DMF with ice-cooling andthe mixture is stirred at room temperature for 4 hours. The mixture ispoured into ice-water, stirred for 30 min and extracted 2× with ethylacetate. The organic phases are washed 2× with water, sat. NaHCO₃solution, 2× with water and brine, dried (Na₂SO₄) and concentrated byevaporation. Column chromatography (SiO₂; methylene chloride/ethanol20:1) and stirring from DIPE yield the title compound: TLC: R_(f)=0.39(methylene chloride/methanol 10:1); HPLC₂₀₋₁₀₀: t_(Ret)=14.0; FAB MS(M+H)⁺=715.

Example 251-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane

With the exclusion of air, 261 mg (1.38 mmol) ofN-methoxycarbonyl-(L)-tert-leucine (Example 2e), 496 mg (2.58 mmol) ofEDC and 232 mg (1.72 mmol) of HOBT are dissolved in 7.5 ml of DMF. After15 min, 0.72 ml (5.17 mmol) of TEA and 585 mg (0.86 mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexanehydrochloride in 3.5 ml of DMF are added. After 20 hours, the mixture isconcentrated by evaporation and water and methylene chloride are addedto the residue; the aqueous phase is separated off and extracted 2× morewith methylene chloride. The organic phases are washed with 10% citricacid solution, sat. NaHCO₃ solution, water and brine, dried (Na₂SO₄) andconcentrated by evaporation. Precipitation from a concentrated solutionin ethyl acetate with DIPE/hexane yields the title compound: HPLC₂₀₋₁₀₀:t_(Ret)=17.5; FAB MS (M+H)⁺=814.

The starting material is prepared as follows:

25a) 4-(Tetrazol-5-yl)-benzaldehyde

20.0 g (0.47 mol) of lithium chloride and 20.5 g (0.315 mol) of sodiumazide are added to 41.2 g (0.315 mol) of 4-cyano-benzaldehyde (Fluka,Buchs, Switzerland) in 310 ml of methoxyethanol (Fluka, Buchs,Switzerland) and the mixture is heated at boiling for 6 hours (argonatmosphere). The cooled reaction mixture is poured into 1 liter ofice/37% HCl 10:1 and stirred thoroughly to complete the reaction.Filtration and washing with water yield the title compound: m.p.:180-182° C.; ¹H-NMR (DMSO-d₆) δ 10.11 (s, HCO), 8.29 and 8.14 (2d, J=8,each 2H).

25b) 4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-benzaldehyde

Under a nitrogen atmosphere, a solution of 6.9 g (58 mmol) of2-phenyl-propene (Fluka, Buchs, Switzerland) and 22 ml of toluene isadded dropwise to 10 g (57 mmol) of 4-(tetrazol-5-yl)-benzaldehyde and 1g (5.7 mmol) of methanesulfonic acid in 44 ml of boiling toluene and themixture is then stirred under reflux conditions for 1 hour. The cooledreaction mixture is washed 2× with sat. NaHCO₃ solution, water andbrine, dried (Na₂SO₄) and concentrated by evaporation to form the titlecompound: ¹H-NMR (DMSO-d₆) δ 10.09 (s, HCO), 8.29 and 8.08 (2d, J=8,each 2H), 7.33 and 7.17 (2m, 5H), 2.17 (s, 6H).

25c)N-1-(tert-Butoxycarbonyl)-N-2-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl-methylidene}-hydrazone

13.0 g (42 mmol) of4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-benzaldehyde and 5.98 g(45.2 mmol) of tert-butyl carbazate in 300 ml of ethanol are stirred at80° C. for 20 hours. The reaction mixture is then concentrated to halfby evaporation; 420 ml of water are added and the mixture is extracted3× with ethyl acetate. The organic phases are washed 2× with sat. NaHCO₃solution and brine, dried (Na₂SO₄) and concentrated by evaporation toform the title compound: HPLC₂₀₋₁₀₀: t_(Ret)=17.7.

25d)N-1-(tert-Butoxycarbonyl)-N-2-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-benzyl}-hydrazine

Under a nitrogen atmosphere, 11.6 g (28.5 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl-methylidene}-hydrazoneare placed in 140 ml of THF, and 2.32 g (31.3 mmol; 85%) of sodiumcyanoborohydride are added. A solution of 5.42 g (28.5 mmol) ofp-toluenesulfonic acid monohydrate in 90 ml of THF is added dropwisethereto. After 4 hours, the mixture is concentrated by evaporation; theresidue is taken up in ethyl acetate and washed with sat. NaHCO₃solution and brine. The aqueous phases are extracted 2× with ethylacetate; the organic phases are dried (Na₂SO₄) and concentrated byevaporation. The residue is taken up in 250 ml of methanol and 125 ml ofTHF, 37 g of K₂B₄O₇ x H₂O in 125 ml of water are added, with cooling,and the mixture is stirred overnight. The mixture is partiallyconcentrated by evaporation using a rotary evaporator and is dilutedwith methylene chloride and water; the aqueous phase is separated offand extracted 2× with methylene chloride. The organic phases are dried(Na₂SO₄) and concentrated by evaporation to form the title compound:HPLC₂₀₋₁₀₀: t_(Ret)=6.4.

25e)1-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-(trifluoroacetyl)amino-6-phenyl-2-azahexane

A mixture of 6.05 g (23.4 mmol) of(2R)-](1′S)-(trifluoroacetyl)amino-2′-phenylethyl]oxirane and 9.54 g(23.4 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-benzyl}-hydrazinein 200 ml of iso-PrOH is heated at 90° C. for 24 hours. Concentration byevaporation, chromatography (SiO₂; methylene chloride/ether 20:1) andcrystallisation from MeOH yield the title compound: Anal. (C₃₄H₄₀N₇O₄F₃)calc. C, 61.16, H, 6.04, N, 14.68: found C, 61.37, H, 6.02, N, 14.80.

25f)1-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane

28 ml of a 1N K₂CO₃ solution are added dropwise, at 70° C., to 1.9 g(2.8 mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}4(S)-hydroxy-2-(tert-butoxycarbonyl)-amino-5(S)-(trifluoroacetyl)amino-6-phenyl-2-azahexanein 29 ml of methanol and the mixture is stirred for 15 hours. Aftercooling and concentration by evaporation, methylene chloride and waterare added; the aqueous phase is separated off and extracted withmethylene chloride. The organic phases are washed with water, dried(Na₂SO₄) and concentrated by evaporation to yield the title compound:HPLC₂₀₋₁₀₀: t_(Ret)=15.1; FAB MS (M+H)⁺=469.

25g)1-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

With the exclusion of air, 868 mg (4.59 mmol) ofN-methoxycarbonyl-(L)-tert-leucine (Example 2e), 1.64 g (8.58 mmol) ofEDC and 773 mg (5.72 mmol) of HOBT are dissolved in 24.5 ml of DMF.After 15 min, 2.39 ml (17.2 mmol) of TEA and 1.64 g (2.86 mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexanein 12 ml of DMF are added. After 20 hours, the mixture is concentratedby evaporation, and water and methylene chloride are added to theresidue; the aqueous phase is separated off and extracted 2× more withmethylene chloride. The organic phases are washed with 10% citric acidsolution, sat. NaHCO₃ solution, water and brine, dried (Na₂SO₄) andconcentrated by evaporation. Digestion from DIPE yields the titlecompound: HPLC₂₀₋₁₀₀: t_(Ret)=18.6; FAB MS (M+H)⁺=743.

25h)1-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexanehydrochloride

Under a nitrogen atmosphere, 1.37 g (1.84 mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexaneare stirred in 64 ml of acetonitrile and 64 ml of aqueous 2N HCl at roomtemperature for 6 days. The reaction mixture is filtered, and thefiltrate is concentrated by evaporation under a high vacuum at roomtemperature and is finally lyophilised from dioxane to yield the titlecompound: HPLC₂₀₋₁₀₀: t_(Ret)=14.2; ¹H-NMR (CD₃OD) inter alia δ 8.10 (d,J=8, 2H^(arom)), 7.8 (m, 1H^(arom)), 7.53 (m, 2H^(arom)), 7.32 (m,3H^(arom)), 7.17 (m, 6H^(arom)), 2.23 (s,2H₃C^(tetrazole-protecting group)).

Example 261-[4-(Tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane

34.5 ml of an 80% aqueous H₂SO₄ solution are added to 345.6 mg (0.424mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane,with ice-cooling. After stirring for 75 min, the mixture is poured into800 ml of ice-water and extracted 3× with ethyl acetate. The organicphases are washed 3× with water and brine, dried (Na₂SO₄) andconcentrated by evaporation. Column chromatography (SiO₂; ethylacetate/ethanol 8:1→2:1) yields the title compound: TLC: R_(f)=0.38(ethyl acetate/ethanol 2:1); HPLC₂₀₋₁₀₀: t_(Ret)=12.5; FAB MS(M+H)⁺=696.

Example 271-[4-(2-Methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane(and 1-methyl-1H-tetrazolyl isomer)

Under a nitrogen atmosphere, 100 mg (0.144 mmol) of1-[4-(tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexaneare dissolved in 1 ml of DMF/dioxane 1:1 and, at 0° C., 73.2 mg (0.224mmol) of CS₂CO₃ and 6.9 μl (0.111 mmol) of methyl iodide in 1 ml ofdioxane are added. The mixture is allowed to warm up slowly to roomtemperature overnight and a further 1 equivalent of Cs₂CO₃ and of methyliodide are added. After stirring for a further 4 hours at roomtemperature, the mixture is diluted with ethyl acetate and 1N sodiumhydroxide solution. The aqueous phase is separated off and extracted 2×with ethyl acetate. The organic phases are washed 2× with water andbrine, dried (Na₂SO₄) and concentrated by evaporation. Columnchromatography (SiO₂; methylene chloride/ethyl acetate 1:1→1:2) yieldsthe pure title compound A (≈3 parts), followed by1-[4-(1-methyl-1H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane(B) (≈1 part): A: TLC: R_(f)=0.26 (methylene chloride/ethyl acetate1:1); HPLC₂₀₋₁₀₀: t_(Ret)=14.2; FAB MS (M+H)⁺=710. B: TLC: R_(f)=0.09(methylene chloride/ethyl acetate 1:1); HPLC₂₀₋₁₀₀: t_(Ret)=13.3; FAB MS(M+H)⁺=710.

Alternative synthesis of the title compound:

Under a nitrogen atmosphere, 14.56 g (77 mmol) ofN-methoxycarbonyl-(L)-tert-leucine and 22.87 g (77 mmol) of TPTU arestirred in 77 ml of DMF and 37.3 ml (218 mmol) of Hünig base at roomtemperature for 30 min. The reaction mixture is then added dropwise toan ice-cooled solution of 35.2 mmol of1-[4-(2-methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanedihydrochloride in 77 ml of DMF. After stirring at room temperature for15 hours, the reaction mixture is partially concentrated by evaporationand the residue (≈80 ml) is poured into 5 liters of water; the mixtureis stirred for 30 min and the crude product is filtered off. Dissolutionin 90 ml of boiling ethanol, addition of 600 ml of DIPE and coolingyield the title compound: m.p.: 191-192° C.; [α]_(D)=−46° (c=0.5;ethanol).

The starting material is prepared as follows:

27a) 4-(2-Methyl-2H-tetrazol-5-yl)-benzaldehyde

With ice-cooling, a solution of 75.5 g (0.434 mol) of4-(tetrazol-5-yl)-benzaldehyde (Example 25a) in 550 ml of DMF/dioxane1:1 is added dropwise to 179.7 g (1.30 mol) of K₂CO₃ in 200 ml ofDMF/dioxane 1:1; the mixture is stirred for 30 min and then 40 ml (0.64mol) of methyl iodide are added. The mixture is stirred in an ice bathfor 3 hours and, finally, at room temperature for 15 hours; the reactionmixture is poured into 2.8 liters of ice-water and stirred for 10 min;the title compound is filtered off and washed with water: m.p.: 137-139°C.; ¹H-NMR (CD₃OD/CDCl₃) d 10.05 (s, HCO), 8.29 and 8.03 (2d, J=8, each2H), 4.43 (s, 3H).

27b)N-1-(tert-Butyloxycarbonyl)-N-2-[4-(2-methyl-2H-tetrazol-5-yl)-phenyl-methylidene]-hydrazone

75.0 g (0.40 mol) of 4-(2-methyl-2H-tetrazol-5-yl)-benzaldehyde and 56.4g (0.426 mol) of tert-butyl carbazate in 1400 ml of iso-PrOH are stirredat 90° C. for 24 hours. 2.2 liters of water are added to the cooledreaction mixture and the mixture is stirred thoroughly to complete thereaction; the title compound is filtered off and washed with water:m.p.: 195-197° C.; Anal. (C₁₄H₁₈N₆O₂) calc. C, 55.62, H, 6.00, N, 27.80:found C, 55.50, H, 5.93, N, 27.61.

27c)N-1-(tert-Butyloxycarbonyl)-N-2-[4-(2-methyl-2H-tetrazol-5-yl)-benzyl]-hydrazine

Under a nitrogen atmosphere, 30.0 g (99.2 mmol) ofN-1-(tert-butyloxycarbonyl)-N-2-[4-(2-methyl-2H-tetrazol-5-yl)-phenyl-methylidene]-hydrazoneare placed in 350 ml of THF, and 8.79 g (119 mmol; 85%) of NaCNBH₃ areadded. A solution of 22.6 g (119 mmol) of p-toluenesulfonic acidmonohydrate in 175 ml of THF is added dropwise thereto (→precipitation). After 2 hours, the solid is filtered off, washedthoroughly with ethyl acetate and discarded. Water and ethyl acetate areadded to the filtrate; the aqueous phase is separated off and extracted2× more with ethyl acetate. The organic phases are washed with sat.NaHCO₃ solution, water and brine, dried (Na₂SO₄) and concentrated byevaporation. The resulting crystals are taken up in 417 ml of methanoland 208 ml of THF, and a solution of 127 g (415 mmol) of K₂B₄O₇.4H₂O in417 ml of H₂O is added dropwise (→ production of foam). The mixture isstirred at room temperature overnight, poured into 2.2 liters of waterand extracted 3× with ethyl acetate. The organic phases are washed withsat. NaHCO₃ solution, water and brine, dried (Na₂SO₄) and concentratedby evaporation. The crude product is combined with material from asecond, identical batch and filtered through silica gel using methylenechloride/THF 10:1 as the eluant. Concentration by evaporation to aresidual volume of about 0.1 liters and addition of 150 ml of DIPE yieldthe crystalline title compound (which, alternatively, may also beobtained by catalytic hydrogenation ofN-1-(Boc)-N-2-[4-(2-methyl-2H-tetrazol-5-yl)-phenyl-methylidene]-hydrazonewith Lindlar catalyst in methanol): m.p.: 100-102° C.; TLC: R_(f)=0.47(methylene chloride/THF 10:1); ¹H-NMR (CD₃OD) d 8.06 and 7.52 (2d, J=8,each 2H), 4.42 (s, 3H); 4.00 (s, 2H); 1.44 (s, 9H); HPLC₂₀₋₁₀₀:t_(Ret)=10.2.

27d)1-[4-(2-Methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butyloxycarbonyl)amino]-6-phenyl-2-azahexane

36.33 g (138 mmol) of (2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane and38.17 g (125 mmol) ofN-1-(tert-butyloxycarbonyl)-N-2-[4-(2-methyl-2H-tetrazol-5-yl)-benzyl]-hydrazineare heated in 964 ml of iso-PrOH at 90° C. for 20 hours. Thecrystallised title compound can be separated from the cooled reactionmixture by filtration. Further product crystallises out of the filtrateafter the addition of 1.2 liters of water: m.p.: 175-178° C.; TLC:R_(f)=0.22 (methylene chloride/ethyl acetate 6:1); HPLC₂₀₋₁₀₀:t_(Ret)=16.9.

27e)1-[4-(2-Methyl-2H-tetrazol-5-yl)-phenyl]-4S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanedihydrochloride

93 ml of 4N aqueous hydrochloric acid solution are added to a solutionof 20.0 g (35.2 mmol) of1-[4-(2-methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butyloxycarbonyl)amino]-6-phenyl-2-azahexanein 279 ml of THF. The mixture is stirred at 50° C. for 8 hours and thenconcentrated gently by evaporation (room temperature; high vacuum). Theoily residue is taken up 3× more in ethanol and again concentrated byevaporation, yielding the crystalline title compound. In order todetermine the analytical data, 1 g of the crude product was stirred in 6ml of hot iso-PrOH, 6 ml of DIPE was added, and cooling and separationby filtration were carried out: m.p.: 227-230° C.; HPLC₂₀₋₁₀₀:t_(Ret)=7.4; Anal. (C₁₉H₂₅N₇O.2HCl (+0.20 H₂O)) calc. C, 51.40, H, 6.22,N, 22.08, Cl, 15.97, H₂O 0.81: found C, 51.50, H, 6.33, N, 22.28, Cl,15.88, H₂O 0.80.

Example 281-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N(N-methoxycarbonyl-(L)-tertleucy)amino-6-phenyl-2-azahexane

With the exclusion of air, 261 mg (1.38 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 496 mg (2.58 mmol) of EDC and 232 mg(1.72 mmol) of HOBT are dissolved in 7.5 ml of DMF. After 15 min, 0.72ml (5.17 mmol) of TEA and 585 mg (0.86 mmol) of1-{4-[2-(1-methyl-1-phenylethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tertleucyl)amino-6-phenyl-2-azahexanehydrochloride (Example 25h) in 3.5 ml of DMF are added. After 20 hours,the mixture is worked up as described under Example 25i. Precipitationwith DIPE from a concentrated solution in methylene chloride yields thetitle compound: HPLC₂₀₋₁₀₀: t_(Ret)=17.5; FAB MS (M+H)⁺=814.

Example 291-[4-(Tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-isoleucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

35 ml of an 80% aqueous H₂SO₄ solution are added to 354 mg (0.435 mmol)of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane,with ice-cooling. After stirring for 75 min, the mixture is worked upanalogously to Example 26 to yield the title compound: HPLC₂₀₋₁₀₀:t_(Ret)=12.6; FAB MS (M+H)⁺=696.

Example 301-[4-(2-Methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 72 mg (0.103 mmol) of1-[4-(tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucylamino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)-amino]-6-phenyl-2-azahexaneare dissolved in 0.5 ml of DMF and, at 0° C., 71 mg (0.217 mmol) ofCs₂CO₃ and 6.9 μl (0.111 mmol) of methyl iodide in 1 ml of dioxane areadded. The mixture is allowed to warm up slowly to room temperatureovernight and is then diluted with ethyl acetate and 1N sodium hydroxidesolution. The aqueous phase is separated off and extracted 2× with ethylacetate. The organic phases are washed 2× with water and brine, dried(Na₂SO₄) and concentrated by evaporation to yield title compound A,which additionally contains ≈20%1-[4-(1-methyl-1H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane(B): HPLC₂₀₋₁₀₀A: t_(Ret)=14.3; HPLC₂₀₋₁₀₀B: t_(Ret)=13.3; FAB MS(M+H)⁺=710.

Example 311-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

With the exclusion of air, 128 mg (0.67 mmol) ofN-methoxycarbonyl-(L)-tert-leucine (Example 2e), 243 mg (1.27 mmol) ofEDC and 114 mg. (0.84 mmol) of HOBT are dissolved in 2 ml of DMF. After15 min, 0.35 ml (2.5mmol) of TEA and 286 mg (0.42 mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanehydrochloride in 1.5 ml of DMF are added. After 20 hours, the mixture isworked up as described under Example 25. Chromatography (SiO₂; ethylacetate/toluene/methylene chloride 2:1:1) yields the title compound:TLC: R_(f)=0.22 (methylene chloride/ethyl acetate 1:1); HPLC₂₀₋₁₀₀:t_(Ret)=17.3; FAB MS (M+H)⁺=814.

The starting materials are prepared as follows:

31a)1-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

With the exclusion of air, 270 mg (1.43 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 513 mg (2.67 mmol) to EDC and 241 mg(1.78 mmol) of HOBT are dissolved in 7.8 ml of DMF. After stirring for15 min, 0.75 ml (5.4 mmol) of TEA and 510 mg (0.89 mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane(Example 25f) in 3.7 ml of DMF are added. After 20 hours, the mixture isworked up analogously to Example 25g to yield the title compound:HPLC₂₀₋₁₀₀: t_(Ret)=18.5; FAB MS (M+H)⁺=743.

31b)1-{4-[2-(1-Methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanehydrochloride

Under a nitrogen atmosphere, 317 mg (0.43 mmol) of1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanein 15 ml of acetonitrile and 15 ml of 2N HCl are stirred at 50° C. for20 hours and worked up analogously to Example 25h to form the titlecompound: HPLC₂₀₋₁₀₀: t_(Ret)=14.4.

Example 321-[4-(Tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tertleucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino]-6-phenyl-2-azahexane

Analogously to Example 26,1-{4-[2-(1-methyl-1-phenyl-ethyl)-2H-tetrazol-5-yl]-phenyl}-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-isoleucyl)amino-6-phenyl-2-azahexaneis deprotected with 80% sulfuric acid to form the title compound.

Example 331-[4-(2-Methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Analogously to Example 30,1-[4-(tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino]-6-phenyl-2-azahexanein DMF/dioxane is methylated with Cs₂CO₃ and methyl iodide.

Example 341-[4-(2-tert-Butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 54 mg (0.28 mmol) ofN-methoxycarbonyl-(L)-tert-leucine and 84 mg (0.28 mmol) of TPTU in 1 mlof DMF and 94 μl (0.85 mmol) of NMM are stirred at room temperature for10 min. 175 mg (0.283 mmol) of1-[4-(2-tert-butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-amino-2-N-[N-methoxycarbonyl-(L)-tert-leucyl]amino-6-phenyl-2-azahexanehydrochloride in 2 ml of DMF are then added thereto and the mixture isstirred at room temperature overnight to complete the reaction. Thereaction mixture is poured into 40 ml of water and extracted 3× withmethylene chloride. The organic phases are filtered through cottonwadding, concentrated by evaporation and chromatographed (SiO₂;methylene chloride/methanol 25:1): TLC: R_(f)=0.48 (methylenechloride/methanol 19:1); HPLC_(20-100(12′)): t_(Ret)=11.8; FAB MS(M+H)⁺=752.

The starting material is prepared as follows:

34a)N-1-(tert-Butoxycarbonyl)-N-2-[N-methoxycarbonyl-(L)-tert-leucyl]-hydrazine

With the exclusion of air, 10.0 g (52.8 mmol) ofN-methoxycarbonyl-(L)-tert-leucine, 11.1 g (58 mmol) of EDC and 7.85 g(58 mmol) of HOBT are placed in 130 ml of ethyl acetate, and 7.0 ml (63mmol) of NMM are added. After 30 min, 7.69 g (58 mmol) of tert-butylcarbazate are added and the mixture is then stirred at room temperaturefor 16 hours. The reaction mixture is diluted with 300 ml of ethylacetate and washed with sat. NaHCO₃ solution, water and brine. Theaqueous phases are back-extracted 2× with ethyl acetate. The organicphases are dried (Na₂SO₄) and concentrated by evaporation to form thetitle compound: ¹H-NMR (CD₃OD) d 3.98 (s, 1H), 3.66 (s, 3H), 1.47 and1.03 (2s, 2×9H).

34b) [N-Methoxycarbonyl-(L)-tert-leucyl]-hydrazine

52.8 mmol ofN-1-(tert-butyloxycarbonyl)-N-2-[N-methoxycarbonyl-(L)-tert-leucyl]-hydrzineare dissolved in 100 ml of 4N HCl/dioxane and stirred at roomtemperature for 18 hours. The suspension is concentrated by evaporation;the residue is taken up in sat. NaHCO₃ solution and extracted 4× withlarge amounts of methylene chloride. Filtration of the organic phasesthrough cotton wadding and concentration by evaporation yield the titlecompound: ¹H-NMR (CD₃OD) d 3.89 (s, 1H), 3.66 (s, 3H), 0.99 (s, 9H).

34c)N-1-[N-Methoxycarbonyl-(L)-tert-leucyl]-N-2-[4-(tetrazol-5-yl)-phenyl-methylidene]-hydrazone

A solution of 3.0 g (14.8 mmol) of[N-methoxycarbonyl-(L)-tert-leucyl]-hydrazine and 2.57 g (14.8 mmol) of4-(tetrazol-5-yl)-benzaldehyde (Example 25a) in 30 ml of isoPrOH isheated at boiling for 18 hours. The mixture is cooled; 100 ml of waterare added and the precipitated title compound is filtered off: ¹H-NMR(CD₃OD) d 8.23 (s, 1H), 8.15-7.9 (m, 4H), 4.08 (s, 1H), 3.67 (s, 3H),1.06 (s, 9H).

34d)N-1-[N-Methoxycarbonyl-(L)-tert-leucyl]-N-2-[4-(2-tert-butyl-2H-tetrazol-5-yl)-phenyl-methylidene]-hydrazone

In an autoclave, 3.0 g (8.3 mmol) ofN-1-[N-methoxycarbonyl-(L)-tert-leucyl]-N-2-[4-(tetrazol-5-yl)-phenyl-methylidene]-hydrazone,1.2 g of iso-butene and 54 μl of methanesulfonic acid in 25 ml oftoluene are heated at 110° C. for 1 hour. The reaction mixture isdiluted with ethyl acetate and washed with sat. NaHCO₃ solution andbrine. The aqueous phases are back-extracted 2× with ethyl acetate; theorganic phases are dried (Na₂SO₄) and concentrated by evaporation.Column chromatography (SiO₂; hexane/ethyl acetate 1:1) yields the titlecompound: TLC: R_(f)=0.22 (hexane/ethyl acetate 1:1);HPLC_(20-100(12′)): t_(Ret)=11.1; FAB MS (M+H)⁺=416.

34e)N-1-[N-Methoxycarbonyl-(L)-tert-leucyl]-N-2-[4-(2-tert-butyl-2H-tetrazol-5-yl)-benzyl]-hydrazine

Under a nitrogen atmosphere, 2.00 g (4.81 mmol) ofN-1-[N-methoxycarbonyl-(L)-tert-leucyl]-N-2-[4-(2-tert-butyl-2H-tetrazol-5-yl)-phenyl-methylidene]-hydrazoneare dissolved in 9 ml of THF, and 317 mg (4.8 mmol; 95%) of NaCNBH₃ areadded. A solution of 915 mg (4.8 mmol) of p-toluenesulfonic acidmonohydrate in 9 ml of THF is added dropwise thereto. After 18 hours,ethyl acetate is added and the mixture is washed with sat. NaHCO₃solution and brine. The aqueous phases are extracted a further 2× withethyl acetate. The organic phases are dried (Na₂SO₄) and concentrated byevaporation. The residue is taken up in 20 ml of THF and 20 ml of water;6.18 g (20 mmol) of K₂B₄O₇.4H₂O are added and the mixture is stirred atroom temperature overnight. The reaction mixture is diluted with ethylacetate and washed with sat. NaHCO₃ solution and brine. The aqueousphases are extracted 2× with ethyl acetate; the organic phases are dried(Na₂SO₄) and concentrated by evaporation. Column chromatography (SiO₂;hexane/ethyl acetate 1:2) yields the title compound: TLC: R_(f)=0.28(hexane/ethyl acetate 1:2); ¹H-NMR (CD₃OD) d 8.07 and 7.53 (2d, J=8,each 2H), 4.03 (s, 2H); 3.84 (s, 1H); 3.64 (s, 3H); 1.81 and 0.92 (2s,each 9H).

34f)1-[4-(2-tert-Butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-N-(tert-butyloxycarbonyl)-amino-2-N-[N-methoxycarbonyl-(L)-tert-leucyl]amino-6-phenyl-2-azahexane

737 mg (2.80 mmol) of (2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane and1.17 g (2.80 mmol) ofN-1-[N-methoxycarbonyl-(L-tert-leucyl]-N-2-[4-(2-tert-butyl-2H-tetrazol-5-yl)-benzyl]-hydrazineare heated in 15 ml of iso-PrOH at 90° C. for 16 hours. On the additionof 100 ml of water the product crystallises and can be filtered off.Recrystallisation by the addition of DIPE/hexane to a concentratedsolution in methylene chloride at 0° C. yields the title compound: TLC:R_(f)=0.34 (CH₂Cl₂/MeOH, 30:1); HPLC_(20-100(12′)): t_(Ret)=12.5.

34g)1-[4-(2-tert-Butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-amino-2-N-[N-methoxycarbonyl-(L)-tert-leucyl]amino-6-phenyl-2-azahexanehydrochloride

Under a nitrogen atmosphere, 200 mg (0.293 mmol) of1-[4-(2-tert-butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-N-(tert-butyloxycarbonyl)amino-2-N-[N-methoxycarbonyl-(L)-tert-leucyl]amino-6-phenyl-2-azahexaneare dissolved in 2.3 ml of THF; 1.6 ml of aqueous 2N HCl are added andthe mixture is stirred at 50° C. for 8 hours. The reaction solution isconcentrated by evaporation; the residue is taken up several times inethanol and concentrated by evaporation again (→title compound): TLC:R_(f)=0.08 (CH₂Cl₂/MeOH 30:1); HPLC_(20-100(12′)): t_(Ret)=9.9; ¹H-NMR(CD₃OD) d 8.03 and 7.50 (2d, J=8, each 2H), 7.32 (m, 5H), 4.18 and 3.91(2d, J=4, 2H), 3.80 (m, 1H), 3.68 (s, 1H), 3.58 (s, 3H), 3.57 (m, 1H),3.3-2.9 (m, 4H), 1.81 and 0.75 (2s, each 9H).

Example 351-[4-(2-tert-Butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 54 mg (0.308 mmol) ofN-methoxycarbonyl-(L)-valine and 92 mg (0.308 mmol) of TPTU in 1 ml ofDMF and 101 μl (0.91 mmol) of NMM are stirred at room temperature for 10min. 190 mg (0.308 mmol) of1-[4-(2-tert-butyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-5(S)-amino-2-N-[N-methoxycarbonyl-(L)-tert-leucyl]amino-6-phenyl-2-azahexanehydrochloride (Example 34g) in 2 ml of DMF are added thereto and themixture is stirred at room temperature overnight to complete thereaction. The reaction mixture is diluted with methylene chloride andwashed with brine. The aqueous phases are extracted 2× with methylenechloride; the organic phases are filtered through cotton wadding,concentrated by evaporation and chromatographed (SiO₂; methylenechloride/methanol 30:1): TLC: R_(f)=0.21 (methylene chloride/methanol19:1); FAB MS (M+H)⁺=738.

Example 361-[4-(2-Methyl-2H-tetrazol-5-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

The title compound may be prepared analogously to one of the Examplesmentioned hereinabove and hereinbelow.

Example 371-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-valyl)amino]-phenyl-2-azahexane

With the exclusion of moisture, 455 mg (2.6 mmol) ofN-methoxycarbonyl-(L)-valine, 940 mg (4.9 mmol) of EDC and 405 mg (3mmol) of HOBT are placed in 10 ml of DMF and heated at 40° C. 1.1 ml(7.9 mmol) of TEA are added and the mixture is stirred for a further 15min. 500 mg (0.98 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanehydrochloride are added thereto and the mixture is stirred at roomtemperature overnight. The reaction mixture is extensively concentratedby evaporation under a high vacuum; the residue is dissolved inmethylene chloride and washed in succession with sodium carbonatesolution (1×), phosphate buffer pH=7 (2×) and brine. After removal ofthe solvent, the residue is chromatographed on silica gel (eluant:methylene chloride/methanol 15:1). The product-containing fractions areconcentrated and the title compound is precipitated with DIPE. Theproduct can be lyophilised from dioxane. HPLC₂₀₋₁₀₀: t_(Ret)=10.06; FABMS (M+H)⁺=677. ¹H-NMR (CD₃OD; 200 MHz) i.a.: 8.58/m (1H); 7.78 and7.50/each d, J=5 (2×2H); 8.0-7.73/m (2H); 7.33/m (1H); 7.30-7.05/m (5H);3.62 and 3.60/each s (2×3H); 1.85 and 1.68/each m (2×1H); 0.76/‘t’, J=4(6H); 0.65 and 0.58/each d, J=4 (2×3H).

The starting material is prepared as follows:

37a) 4-Bromobenzaldehyde dimethyl acetal

21.1 g (114 mmol) of 4-bromobenzaldehyde and 20 ml (182 mmol) oftrimethyl orthoformate (both Fluka, Buchs, Switzerland) are dissolved in35 ml of methanol, and 0.65 g (3.4 mmol) of p-toluenesulfonic acidmonohydrate is added at room temperature (exothermic reaction). Thereaction mixture is stirred at room temperature under nitrogen for 20hours. The acid is then neutralised with 0.62 ml of 30% sodiummethanolate solution in methanol (3.4 mmol); the reaction mixture isconcentrated using a rotary evaporator and the residue is distilled. Thetitle compound is obtained in the form of a colourless liquid. TLC:R_(f)=0.58 (hexane/ethyl acetate 2:1). B.p.: 90-92° C. (4 mbar). 1H-NMR(CDCl₃; 200 MHz): 7.50 and 7.32/each d, J=9 (2×2H); 5.36/s (1H); 3.31/s(6H).

37b) 4-(Pyridin-2-yl)-benzaldehyde

6.93 g (29.9 mmol) of 4-bromobenzaldehyde dimethyl acetal in 40 ml ofTHF are added dropwise to a warm (from 40° C. to 50° C.) suspension of0.8 g (31.6 mmol) of magnesium turnings and a small amount of iodine in10 ml of THF. The reaction mixture is heated to 65° C. and stirred atthat temperature for about 30 min. The mixture is allowed to cool toroom temperature and the Grignard reagent is added dropwise to asolution of 4.46 g (28.2 mmol) of 2-bromopyridine (Fluka, Buchs,Switzerland) and 0.4 g (0.74 mmol) of DPPP (Fluka, Buchs, Switzerland)in 100 ml of THF (slightly exothermic reaction). After the dropwiseaddition is complete, the reaction mixture is boiled under reflux for 4hours and is then allowed to cool; 100 ml of water are added. Themixture is concentrated to about 50 ml using a rotary evaporator,diluted with ethyl acetate and extracted with 0.1N hydrochloric acid(3×). The combined HCl extracts are stirred at room temperature for 20min, rendered basic with concentrated ammonia solution and extractedwith methylene chloride. After removal of the solvent, the residue ischromatographed on silica gel (hexane/ethyl acetate 2:1). Theproduct-containing fractions are concentrated, with the desired titlecompound crystallising out spontaneously. TLC: R_(f)=0.22 (hexane/ethylacetate 2:1). HPLC₂₀₋₁₀₀: t_(Ret)=6.08. 1H-NMR (CDCl₃; 200 MHz): 8.73/d,J=5 (2H); 8.16 and 7.97/each d (2×2H); 7.80/d, J=4 (2H); 7.3/m (1H).

37c)N-1-(tert-Butoxycarbonyl)-N-2-{4-[(pridin-2-yl)-phenyl]-methylidene}-hydrazone

A solution of 2 g (1.05 mmol) of 4-(pyridin-2-yl)-benzaldehyde and 1.37g (1 mmol) of tert-butyl carbazate (Fluka, Buchs, Switzerland) in 30 mlof ethanol is stirred at 80° C. for 5 hours (after 4 hours, a further0.05 equivalent of tert-butyl carbazate is added). The reaction mixtureis allowed to cool and diluted with water, with the desired titlecompound crystallising out TLC: R_(f)=0.51 (methylene chloride/methanol15:1). HPLC₂₀₋₁₀₀: t_(Ret)=8.92. 1H-NMR (CDCl3; 200 MHz): 8.68/m (1H);8.21/s (1H); 7,98/d, J=9 (2H, portion A of aromatic AB system); 7.85/s(1H); 7.8-7.6/m (4H); 7.22/m (1H); 1.53/s (9H).

37d) N-1-(tert-Butoxycarbonyl)-N-2-[4(pyridin-2yl)-benzyl]-hydrazine

2 g (6.7 mmol) ofN-1-(tert-butoxcarbonyl)-N-2-{4-[(pyridin-2-yl)-phenyl]-methylidene}-hydrazoneand 0.2 g of 5% Pd/C in 30 ml of methanol are hydrogenated under normalpressure at room temperature for 8 hours. The catalyst is filtered offand washed with methanol; the solvent is removed. The title compound isobtained in the form of a colourless, viscous oil, which solidifies ondrying under a high vacuum. TLC: R_(f)=0.46 (metylene chloride/methanol15:1). HPLC₂₀₋₁₀₀: t_(Ret)=6.71. 1H-NMR (CDCl₃; 200 MHz) i.a.: 8.69/m(1H), 7.96 and 7.45/each d, J=2 (2×2H); 7.8-7.65/m (2H); 7.22/m (1H);4.06/s (2H); 1.47/s (9H).

37e)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexane

A solution of 1.06 g (4 mmol) of(2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane and 1.2 g (4 mmol ofN-1-(tert-butoxycarbonyl)-N-2-[4-(pyridin-2-yl)-benzyl]-hydrazine in 20ml of iso-PrOH is stirred at 80° C. for 16 hours. After cooling, thereaction solution is concentrated using a rotary evaporator, with thetitle compound precipitating out as a colourless precipitate. Furtherproduct can be precipitated out by adding water to the mother liquor.TLC: R_(f)=0.53 (methylene chloride/methanol 15:1). HPLC₂₀₋₁₀₀.t_(Ret)=13.15. 1H-NMR (CD₃OD; 200 MHz) i.a.: 8.57/s (1H); 7.85 and7.48/each d, J=9 (2×2H); 8.0-7.7/m (2H); 7.33/m (1H); 7.3-7.0/m (6H);3.91/s (2H),; 3.82-3.55/m (2H); 3.05-2.45/m (4H); 1.31/s (18H).

37h)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanehydrochloride

10 ml of DMF are added to a mixture consisting of 1.43 g (2.54 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azhexanein 30 ml of 4N hydrogen chloride in dioxane (Aldrich) (exothermicreaction) and the mixture is stirred at room temperature for 2 hours.The solvent is then removed; toluene is added to the residue three timesand the mixture is concentrated by evaporation. The residue is dissolvedin hot methanol and the title compound is precipitated in the form of aresinous precipitate with DIPE/hexane. On drying under a high vacuum, avoluminous foam is obtained. HPLC₅₋₆₀: t_(Ret)=9.87. 1H-NMR (CD₃OD; 200MHz) i.a.: 8.78/d, J=5 (1H); 8.72/dxt, J=2.5 and 7.5 (1H); 8.35/d, J=7.5(1H); 8.1/d×d, J=each 7.5 (1H); 8.02 and 7.72/each d, J=9 (2×2H);7.45-7.15/m (5H); 4.27 and 4.15/each d, J=12.5 (2×2H).

Example 381-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-ethoxycarbonyl-L)-valyl)amino]-6-phenyl-2-azahexane

Analogously to Example 37, after working up the title compound isobtained from 300 mg (0.59 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanehydrochloride (Example 37f), 446 mg (2.36 mmol) ofN-ethoxycarbonyl-(L)-valine, 679 mg (3.54 mmol) of EDC, 398 mg (2.95mmol) of HOBT and 0.82 ml (5.9 mmol) of TEA in 10 ml of DMF. TLC:R_(f)=0.19 (methylene chloride/methanol 15:1). HPLC₂₀₋₁₀₀:t_(Ret)=11.68. FAB MS (M+H)⁺=705.

Example 391-[4-(Pyridin-3-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-valyl)amino]-phenyl-2-azahexane

Analogously to Example 37, the title compound is obtained from 550 mg(1.52 mmol) of1-[4-(pyridin-3-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexane691 mg (3.94 mmol) of N-methoxycarbonyl-(L)-valine, 1.45 g (7.59 mmol)of EDC, 614 mg (4.55 mmol) of HOBT and 1.06 ml (7.59 mmol) of TEA in 10ml of DMF. (Contrary to the description in Example 37, the organic phaseis washed with sat. sodium hydrogen carbonate solution, 10% citric acidand brine.) TLC: R_(f)=0.4 (methylene chloride/methanol 15:1).HPLC₂₀₋₁₀₀: t_(Ret)=9.91; FAB MS (M+H)⁺=677.

The starting material is prepared as follows:

39a) 4-(Pyridin-3-yl)-benzaldehyde

Analogously to Example 37b, the title compound is obtained from 6.39 g(29.9 mmol) of 4-bromobenzaldehyde dimethyl acetal (prepared inaccordance with Example 37a), 0.8 g (31.6 mmol) of magnesium turnings,2.77 ml (28.2 mmol) of 3-bromopyridine (Fluka, Buchs, Switzerland) and0.4 g (0.74 mmol) of DPPP in 150 ml of THF. HPLC₂₀₋₁₀₀: t_(Ret)=5.50. 1HNMR (CD₃OD; 200 MHz): 10.04/s (1H); 8.87/d, J=2.5 (1H); 8.58/d×d,J=about 1.5 and 5 (1H); 8.17/m i.a. J=7.5 (1H); 8.05 and 7.88/each d,J=9 (2×2H); 7.56/d×d, J=7.5 and 5 (1H).

39b)N-1-(tert-Butoxycarbonyl)-N-2-{4-[(pyridin-3-yl)-phenyl]-methylidene}-hydrazone

Analogously to Example 37c, the title compound is obtained from 4.11 g(22.4 mmol) of 4-(pyridin-3-yl)-benzaldehyde and 2.82 g (21.3 mmol) oftert-butyl carbazate (Fluka, Buchs, Switzerland) in 60 ml of ethanol.HPLC₂₀₋₁₀₀: t_(Ret)=8.88. 1H-NMR (CD₃OD; 200 MHz): 8.83/d, J=2.5 (1H);8.53/d, J=5 (1H); 8.14/m i.a. J=7.5 (1H); 7.97/s (1H); 7.97/s (1H); 7.85and 7.71/each d, J=9 (2×2H); 7.53/d×d, J=7.5 and 5 (1H).

39c) N-1-(tert-Butoxycarbonyl)-N-2-[4-(pyridin-3-yl)-benzyl]-hydrazine

Analogously to Example 37d, the title compound is obtained from 5.03 g(16.9 mmol) ofN-1-(tert-butoycarbonyl)-N-2-{4-[(pyridin-3-yl)-phenyl]-methylidene}-hydrazoneand 0.5 g of 5% Pd/C in 120 ml of methanol, the title compound beingprocessed further in unpurified form. HPLC₂₀₋₁₀₀: t_(Ret)=6.36. 1H-NMR(CD₃OD; 200 MHz) i.a.: 7.63 and 7.51/each d, J=9 (2×2H); 3.97/s (2H);1.43/s (9H).

39d)1-[4-(Pyridin-3-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexane

Analogously to Example 37e, the title compound is obtained from 3.82 g(12.8 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(pyridin-3-yl)-benzyl]-hydrazine and3.36 g (12.8 mmol) of (2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane after14 hours at 80° C. Purification is carried out by chromatography onsilica gel (hexanelethyl acetate 1:2). TLC: R_(f)=0.27 (hexane/ethylacetate 1:2). HPLC₂₀₋₁₀₀: t_(Ret)=13.0. 1H-NMR (CD₃OD; 200 MHz) i.a.:7.62 and 7.52/each d, J=9 (2×2H); 7.4-7.0/m (5H); 3.93/s (2H); 1.33 and1.31/each s (2×9H).

39e)1-[4-(Pyridin-3-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexane

1 g (1.88 mmol) of1-[4-(pyridin-3-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexaneis dissolved in 10 ml of formic acid and the solution is stirred at roomtemperature for 5 hours. The reaction mixture is concentrated byevaporation, the residue dissolved in methylene chloride and the organicphase washed with sat. sodium hydrogen carbonate solution and brine.After removal of the solvent, the title compound is obtained in the formof a brown oil, which is processed further without purification.

Example 401-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-valyl)amino]phenyl-2-azahexane

Analogously to Example 37, the title compound is obtained from 473 mg(0.75 mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanehydrochloride, 263 mg (1.5 mmol) of N-methoxycarbonyl-(L)-valine, 575 mg(3 mmol) of EDC (Fluka, Buchs, Switzerland), 405 mg (3 mmol) of HOBT(Fluka) and 1.7 ml (12 mmol) of TEA in 10 ml of DMF. Working up isperformed analogously to Example 40f, using ethyl acetate instead ofmethylene chloride. The compound can be lyophilised from dioxane. TLC:R_(f)=0.28 (ethyl acetate). HPLC₂₀₋₁₀₀: t_(Ret)=13.11; FAB MS(M+H)⁺=678.

The starting material is prepared as follows:

40a) 4-(Pyrazin-2-yl)-benzaldehyde

[see EP 0 344 577]

50 ml of THF are poured over 2.72 g (112 mmol) of magnesium turnings,which have been de-greased with hexane and activated with a small amountof iodine, and the mixture is heated at 50° C. A solution of4-bromobenzaldehyde dimethyl acetal (prepared in accordance with Example37a) in 200 ml of THF is added dropwise to the mixture within a periodof about 30 min. Initially, the reaction is exothermic; towards the endof the dropwise addition the reaction mixture is heated to about 60° C.After stirring at 60° C. for a further 30 min, the mixture is allowed tocool to room temperature and decanted off from the unreacted magnesium;the resulting solution containing the Grignard reagent is added dropwiseat room temperature over a period of 20 min to a suspension of 11.45 g(100 mmol) of 2-chloropyrazine (Fluka, Buchs, Switzerland) and 1.6 g ofDPPP (Aldrich, Buchs, Switzerland) in 500 ml of THF (slightly exothermicreaction). The mixture is then stirred at room temperature for 19 hours.Then 250 ml of water are added to the reaction mixture and the mixtureis stirred for 10 min. The THF is removed in vacuo; 300 ml of ethylacetate and 100 ml of 2N hydrochloric acid are added to the emulsionthat remains and the mixture is stirred for 5 min. After separation ofthe organic phase, that phase is stirred twice more with 100 ml, in eachcase, of 0.5N hydrochloric acid for 5 min. The ethyl acetate phase iswashed in succession with sat. sodium hydrogen carbonate solution, waterand brine and is concentrated. The title compound is obtained in theform of light-brown crystals. Recrystallisation from methylenechloridelhexane is carried out. M.p.: 86-88° C. TLC: R_(f)=0.17(hexane/ethyl acetate 2:1). HPLC₂₀₋₁₀₀: t_(Ret)=1.06. 1H-NMR (CDCl₃; 200MHz): 10.12/s (1H); 9.14/d, J≦1 (1H); 8.70/d, J≦1 (1H); 8.60/t, J≦1(1H); 8.22 and 8.03/each d, J=9 (2×2H).

40b)N-1-(tert-Butoxycarbonyl)-N-2-{4-[(pyrazin-2-yl)-phenyl]-methylidene}-hydrazone

Analogously to Example 37c, the title compound is obtained from 12.4 g(67.3 mmol) of 4-(pyrazin-2-yl)-benzaldehyde and 8.5 g (64 mmol) oftert-butyl carbazate (Fluka, Buchs, Switzerland) in 170 ml of ethanolafter 5 hours at 80° C., with the title compound crystallising outspontaneously. M.p: 190-198° C. TLC: R_(f)=0.47 (ethyl acetate).HPLC₂₀₋₁₀₀: t_(Ret)=13.41.

40c) N-1-(tert-Butoxycarbonyl)-N-2-[4-(pyrazin-2-yl)-benzyl]-hydrazine

Analogously to Example 37d, the title compound is obtained in the formof an oil from 0.6 g (2 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-{4-[(pyrazin-2-yl)-phenyl]-methylidene}-hydrazoneand 0.15 g of 5% Pd/C in 15 ml of THF after hydrogenation for 13 hoursat room temperature. The title compound crystallises out on triturationwith ether. Recrystallisation from ethyl acetate/petroleum ether iscarried out. M.p.: 110-111° C. HPLC₂₀₋₁₀₀: t_(Ret)=9.62. 1H-NMR (CD₃OD;200 MHz): 9.09/s (1H); 8.65/t, J≦1 (1H); 8.51/t, J≦1 (1H); 8.05 and7.53/each d, J=5 (2×2H); 4.00/s (2H); 1.43/s (9H).

40d)1-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-(trifluoroacetyl)amino-6-phenyl-2-azadhexane

Analogously to Example 37e, the title compound is obtained in the formof beige crystals from 10.5 g (35 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(pyrazin-2-yl)-benzyl]-hydrazine and11.7 g (45 mmol) of(2R)-[(1′S)-(trifluoroacetyl)amino-2′-phenylethyl]oxirane (EP 0 521 827,Example 16d) in 150 ml of isopropanol. M.p.: 194-196° C. TLC: R_(f)=0.38(hexane/ethyl acetate 1:2). HPLC₂₀₋₁₀₀: t_(Ret)=16.27.

40e)1-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane

11.75 g (21 mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-(trifluoroacetyl)amino-6-phenyl-2-azahexaneare suspended in 500 ml of methanol and, at 60° C., 105 ml of a 1M K₂CO₃solution in water are added. The mixture is stirred at 75° C. for about3 hours; the methanol is evaporated off and the residue is extractedwith ethyl acetate. The organic phase is washed once each with water andbrine and concentrated. The title compound is obtained in the form oforange-brown crystals, which can be recrystallised from ethylacetate/petroleum ether. M.p.: 146-148° C. TLC: R_(f)=0.08 (methylenechloride/methanol 10:1). HPLC₂₀₋₁₀₀: t_(Ret)=11.23.

40f)1-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Analogously to Example 37, the title compound is obtained from 3.2 g (7mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane,2.54 g (14 mmol) of N-methoxycarbonyl-(L)-valine, 5.4 g (28 mmol) of EDC(Fluka, Buchs, Switzerland), 3.8 g (28 mmol) of HOBT (Fluka, Buchs,Switzerland) and 7.1 g (70 mmol) of TEA in 130 ml of DMF. The reactionmixture is worked up by removing the DMF, taking up the residue inmethylene chloride and washing the organic phase in succession withwater, sat. sodium hydrogen carbonate solution/water 1:1, 10% citricacid, water and brine. The compound crystallises out on concentration.M.p.: 218-220° C. TLC: R_(f)=0.29 (methylene chloride/methanol 10:1).HPLC₂₀₋₁₀₀: t_(Ret)=15.11.

40g)1-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanehydrochloride

3.4 g (5.5 mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanein 100 ml of 4N hydrogen chloride in dioxane. (Aldrich) and 10 ml ofmethanol are stirred at room temperature for 2 hours. The solvents areremoved; dioxane is added twice to the residue and evaporated off. Thetitle compound is obtained in the form of a viscous oil, with thecompound crystallising out on trituration with ether. M.p.: 194-198° C.TLC: R_(f)=0.35 (methylene chloride/methanol 10:1). HPLC₂₀₋₁₀₀:tRet^(9.77.)

Example 411-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-[N-(N-methoxycarbonyl-(L)-iso-leucyl)amino]-5(S)-[N-(N-methoxycarbonyl-(L)-valyl)amino]-6-phenyl-2-azahexane

142 mg (0.75 mmol) of N-methoxycarbonyl-(L)-iso-leucine and 223 mg (0.75mmol) of TPTU in 3 ml of DMF are stirred at room temperature for 10 minand then a solution of 473 mg (0.75 mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanehydrochloride (Example 40g) and 0.33 ml of NMM in 3 ml of DMF is added.The mixture is stirred at room temperature overnight. Working up iscarried out by the slow, dropwise addition of the reaction mixture to100 ml of water, stirring at room temperature for 20 min and isolationof the resulting precipitate by filtration. The precipitate is washedwith water and taken up in methylene chloride; the organic phase iswashed in succession with water, sat. sodium hydrogen carbonatesolution/water 1:1, water and brine. After removal of the solvent, theresidue is digested in ether, with the title compound being obtained inthe form of a colourless powder. The compound can be lyophilised fromdioxane. TLC: R_(f)=0.28 (ethyl acetate). HPLC₂₀₋₁₀₀: t_(Ret)=13.78. FABMS(M+H)⁺=692.

Example 421-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-5(S)-[N-(N-methoxycarbonyl-(L)-valyl)amino]-6-phenyl-2-azahexane

Analogously to Example 41, after working up the title compound isobtained from 142 mg (0.75 mmol) of N-methoxycarbonyl-(L)-tert-leucine(Example 2e), 223 mg (0.75 mmol) of TPTU in 3 ml of DMF (solution A) and435 mg (0.75 mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanehydrochloride (Example 40g) and 0.33 ml of NMM in 3 ml of DMF (solutionB), the title compound crystallising out spontaneously on evaporation ofthe solvent. The compound can be lyophilised from dioxane. TLC:R_(f)=0.46 (methylene chloride/methanol 10:1). HPLC₂₀₋₁₀₀:t_(Ret)=13.85. FAB MS(M+H)⁺=692.

Example 431-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-iso-leucyl)amino]-6-phenyl-2-azahexane

Analogously to Example 41, after working up the title compound isobtained from 132 mg (0.7 mmol) of N-methoxycarbonyl-(L)-iso-leucine and208 mg (0.7 mmol) of TPTU in 3 ml of DMF (solution A) and 400 mg (0.7mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanehydrochloride (Example 44b) and 0.31 ml (2.8 mmol) of NMM in 3 ml ofDMF, the title compound being obtained in crystalline form by digestionwith ether. M.p.: 211-217° C. TLC: R_(f)=0.41 (methylenechloride/methanol 10:1). HPLC₂₀₋₁₀₀: t_(Ret)=14.49. FAB MS(M+H)⁺=706.

Example 441-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-[N-(N-methoxycarbonyl-(L)-valyl)amino]-5(S)-[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane

Analogously to Example 41, after working up the title compound isobtained from 175 mg (1 mmol) of N-methoxycarbonyl-(L)-valine, 297 mg (1mmol) of TPTU (Fluka, Buchs, Switzerland) in 4 ml of DMF (solution A)and 571 mg (1 mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanehydrochloride and 0.44 ml (4 mmol) of NMM in 4 ml of DMF (solution B);the title compound can be obtained in crystalline form by digestion withether. M.p.: 205-208° C. HPLC₂₀₋₁₀₀: t_(Ret)=13.87. FAB MS(M+H)⁺=692.

The starting material is prepared as follows:

44a)1-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Analogously to Example 37, the title compound is obtained from 2.3 g (5mmol) of1-[4-(pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-amino-6-phenyl-2-azahexane(Example 40e), 1.9 g (10 mmol) of N-methoxycarbonyl-(L)-iso-leucine, 3.8g (20 mmol) of EDC, 2.7 g (20 mmol) of HOBT and 5.1 g (50 mmol) of TEAin 90 ml of DMF. Working up is carried out as described in Example 40f.The compound can be recrystallised from ethyl acetate. TLC: R_(f)=0.58(methylene chloride/methanol 10:1). HPLC₂₀₋₁₀₀: t_(Ret)=15.68. 1H-NMR(CD₃OD; 200 MHz) i.a.: 9.08/s (1H); 8.65/bs (1H); 8.51/t, J≦1 (1H); 8.02and 7.52/each d, J=5 (2×2H); 7.3-7.1/m (5H); 3.92/s (2H); 3.62/s (3H);1.28/s (9H); 0.8/t, J=5 (3H); 0.73/d, J=4 (3H).

44b)1-[4-(Pyrazin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanehydrochloride

Analogously to Example 40g, the title compound is obtained from 2.1 g(3.3 mmol) of1-[4-(pyrazin-2-yl)-pheny]-4(S)-hydroxy-2-(tert-butoxycarbonyl)amino-5(S)-N-(N-methoxy-carbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanein 60 ml of 4N hydrogen chloride in dioxane and 10 ml of methanol, withthe title compound being caused to crystallise with ether. M.p.:200-201° C. HPLC₂₀₋₁₀₀: t_(Ret)=10.52.

Example 451-[4-(Thiophen-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxy-carbonyl-(L)-valyl)amino]-6-phenyl-2-azahexane

Analogously to Example 37, the title compound is obtained from 500 mg(1.36 mmol) of1-[4-(thiophen-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexane,620 mg (3.54 mmol) of N-methoxycarbonyl-(L)-valine, 1.3 g (6.8 mmol) ofEDC, 551 mg (4.08 mmol) of HOBT and 3.95 ml (6.8 mmol) of TEA in 10 mlof DMF, the title compound being lyophilised from dioxane. TLC:R_(f)=0.51 (methylene chloride/methanol 15:1). HPLC₂₀₋₁₀₀:t_(Ret)=15.30. FAB MS(M+H)⁺=682.

The starting material is prepared as follows:

45a) 4-(Thiophen-2-yl)-benzaldehyde

[see Heterocycles 31, 1951 (1990)]

3.7 g (20 mmol) of 4-bromobenzaldehyde, 9.5 ml (120 mmol) of thiophene,2.94 g (30 mmol) of potassium acetate and 1.16 g (1 mmol) oftetrakis(triphenylphosphine)-palladium (Fluka, Buchs, Switzerland) in 50ml of dimethylacetamide are placed in a pressure reactor and stirred at150° C. under nitrogen for 16 hours. The reaction mixture isconcentrated by evaporation; the residue is taken up in water andextracted three times with methylene chloride. After removal of thesolvent, the residue is chromatographed on silica gel (hexanelethylacetate 4:1). The title compound is obtained in the form of a yellowsolid. TLC: R_(f)=0.36 (hexane/ethyl acetate 4:1). HPLC₂₀₋₁₀₀:t_(Ret)=15.26. 1H-NMR (CD₃OD; 200 MHz): 9.98/s (1H); 7.93 and 7.85/eachd, J=9.5 (2×2H); 7.60/d, J=2.5 (1H); (1H); 7.17/d×d, J=2.5 and 5 (1H).

45b)N-1-(tert-Butoxycarbonyl)-N-2-{4-[(thiophen-2-yl)-phenyl]-methylidene}-hydrazone

Analogously to Example 37c, the title compound is obtained in the formof yellow crystals from 2.47 g (13.1 mmol) of4-(thiophen-2-yl)-benzaldehyde and 1.65 g (12.49 mmol) of tert-butylcarbazate (Fluka, Buchs, Switzerland) in 30 ml of ethanol (4.5 hours at90° C.). M.p.: 162-165° C. HPLC₂₀₋₁₀₀: t_(Ret)=16.08. 1H-NMR (CD₃OD; 200MHz) 1.53/s (9H).

45c) N-1-(tert-Butoxycarbonyl)-N-2-[4-(thiophen-2-yl-benzyl]-hydrazine

3.35 g (11.1 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-{4-[(thiophen-2-yl)-phenyl]-methylidene}-hydrazoneand 0.819 g (11.1 mmol) of sodium cyanoborohydride (Fluka, Buchs,Switzerland) are dissolved in 11 ml of THF (black solution) and addeddropwise over a period of 5 hours to 2.11 g (11.1 mmol) ofp-toluenesulfonic acid monohydrate dissolved in 11 ml of THF. Themixture is stirred overnight at room temperature and under nitrogen(pH=about from 3 to 4) and then diluted with ethyl acetate; the organicphase is washed in succession with brine, sat. sodium hydrogen carbonatesolution and again brine. The organic phase is concentrated byevaporation and the residue is taken up in 13.3 ml of 1N sodiumhydroxide solution; 15 ml of methylene chloride are added and themixture is boiled under reflux for 3 hours at a bath temperature of 60°C. After separation of the organic phase, that phase is concentrated todryness by evaporation. The title compound is obtained in the form of aslightly yellowish oil. HPLC₂₀₋₁₀₀: t_(Ret)=12.36. 1H-NMR (CD₃OD; 200MHz) i.a.: 3.91/s (2H); 1.42/s (9H).

45d)1-[4-(Thiophen-2-yl-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)-amino]-6-phenyl-2-azahexane

Analogously to Example 37e, the title compound is obtained from 3.39 g(11.1 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(thiophen-2-yl)-benzyl]-hydrazine and2.93 g (11.1 mmol) of (2R)-[(1′S)-Boc-amino-2′-phenylethyl]oxirane (J.Org. Chem. 50, 4615 (1985)) in 50 ml of isopropanol, with the titlecompound crystallising out spontaneously on cooling of the reactionsolution. M.p.: 165-168° C. HPLC₂₀₋₁₀₀: t_(Ret)=18.84. 1H-NMR (CD₃OD;200 MHz) i.a.: 7.56/d, J=9 (2H); 7.5-7.3/m (4H); 7.3-7.1/m (5H);7.08/d×d, J=2 and 5 (1H); 3.85/s (2H); 1.33 and 1.32/each s (2×9H).

45e)1-[4-Thlophen-2-yl)-phenyl]-4S-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexane

Analogously to Example 39e, the title compound is obtained in the formof a slightly yellowish oil from 3.16 g (5.57 mmol) of1-[4-(thiophen-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexanein 30 ml of formic acid after stirring at room temperature for 6 hours,that oil, being processed further without purification. 1H-NMR (CD₃OD;200 MHz) i.a.: 7.62/d, J=9 (2H); 7.5-7.1/several m's, superimposed (9H);7.09/d×d, J=2 and 5 (1H); 3.72/s (2H).

Example 461-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane

Process A:

With the exclusion of moisture, 10.85 g ofN-methoxycarbonyl-(L)-tert-leucine (Example 2e) and 17.1 g of TPTU areplaced in 65 ml of DMF. 35.1 ml of Hünig base are added to the whitesuspension and the mixture is stirred at room temperature for 20 min.Then 13.2 g (26 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanehydrochloride (Example 37f) dissolved in 65 ml of DMF are added and themixture is stirred for 24 hours to complete the reaction (after 20hours, a further 5 ml of Hünig base are added). The reaction mixture ispoured into 600 ml of water and the resulting precipitate is filteredoff and washed with water. The filter residue is then dissolved inmethylene chloride and washed 2× with sat. NaHCO₃ solution, water andbrine. After drying over sodium sulfate and concentration, the resultingfoam is digested with DIPE; the solid is filtered off and dried. Theresulting crude product is dissolved again in methylene chloride,treated with active carbon and, after filtration, precipitated withether. The resulting title compound is dried in a heated desiccator at40° C. under a high vacuum: m.p.: 202-204° C.; TLC: R_(f)=0.38 (ethylacetate); HPLC₂₀₋₁₀₀: t_(Ret)=11.81; FAB MS (M+H)⁺=705. Further productcan be obtained from the mother liquor after chromatography (SiO₂,hexane/ethyl acetate, then ethyl acetate) and after crystallisation fromether (m.p. 206-207° C.).

Process B:

Analogously to Example 4, 1.32 g of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexanein 5 ml of DMF are added to 0.42 g (2.2 mmol) of(N-methoxycarbonyl-(L)-tert-leucine, 0.654 g (2.2 mmol) of TPTU and 840μl (5 mmol) of Hünig Base in 5 ml of DMF, and the mixture is stirred atroom temperature for 22 hours and worked up analogously to Example 3 toyield the title compound.

The starting compounds are prepared as follows:

46a)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-Boc-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

Analogously to Example 1, a solution of 3.93 g (8.5 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-(N-Boc-amino)-5(S)-amino-6-phenyl-2-azahexanehydrochloride (Example 47b) in 50 ml of DMF is added dropwise to amixture of 2.58 g (13.6 mmol) of N-methoxycarbonyl-(L)-tert-leucine,4.88 g (25.5 mmol) of EDC and 2.3 g (17 mmol) of HOBT in 50 ml of DMF.After working up, the crude product is digested in methylenechloride/DIPE, filtered off and dried to yield the title compound. TLC:R_(f)=0.5 (ethyl acetate); HPLC₂₀₋₁₀₀: t_(Ret)=12.32; FAB MS (M+H)⁺=634.

46b)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexanehydrochloride

Analogously to Example 37f), 130 ml of 4M HCl in dioxane are added to4.4 g (6.94 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-Boc-amino-5(S)-N-(N-methoxyarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexaneand the mixture is diluted with 7 ml of DMF. After 2.75 hours, themixture is worked up. The title compound is obtained: TLC: R_(f)=0.44(methylene chloride/methanol: 9/1); HPLC₂₀₋₁₀₀: t_(Ret)=8.47; FAB MS(M+H)⁺534.

An alternative procedure for the preparation of the title compound fromExample 46 is as follows:

Example 46*1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-phenyl-2-azahexane

With the exclusion of moisture, 567 g (3.0 mol) ofN-methoxycarbonyl-(L)-tert-leucine (Example 2e) and 891 g (3.0 mol) ofTPTU are placed in 3 liters of methylene chloride; with ice-cooling, 775g (6 mol) of Hünig base are added dropwise and the mixture is stirredfor 20 min. A suspension of 432 g (1.0 mol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanetrihydrochloride in 3 liters of methylene chloride is then added to thesolution and the mixture is stirred at room temperature overnight tocomplete the reaction. The reaction mixture is washed with 10 liters ofwater, 10 liters of sat. NaHCO₃ solution and 5 liters of brine. Theaqueous phases are extracted a further 2× with 5 liters of methylenechloride; the organic phases are dried (Na₂SO₄) and concentrated byevaporation. The residue is dissolved in 6 liters of ethyl acetate andfiltered through 500 g of silica gel; the column is rinsed with 6 litersof ethyl acetate and the product-containing fractions are concentratedby evaporation. Stirring in boiling DIPE/ethanol 49:1 (9 liters; 1hour), cooling and filtration yield the title compound, which can befurther purified by recrystallisation from ethanol/water (m.p. 207-209°C.).

The starting compounds are prepared as follows:

*a) 4-(Pyridin-2-yl)-benzaldehyde

11 g of iodine, followed by 200 g of 4-bromobenzaldehyde dimethyl acetal(Example 37a), are added to 317 g (13.0 mol) of magnesium in 3.5 litersof THF (nitrogen atmosphere). Once the reaction has started (heating ifnecessary), 2540 g (in total 2740 g; 11.8 mol) of 4-bromobenzaldehydedimethyl acetal in 3.5 liters of toluene are added dropwise (from 25° to30° C., 1 hour) and the mixture is then stirred at room temperature for1 hour. The Grignard reagent is then transferred to the dropping funnelof a second apparatus containing 1750 g (11.0 mol) of 2-bromopyridine(Fluka, Buchs, Switzerland) in 3.3 liters of THF, 38 g (70 mmol) of DPPPand 330 ml of diisobutylaluminium hydride (20% in hexane). At from 150to 20° C., the Grignard reagent is added dropwise (45 min). After beingstirred at room temperature for 90 min, the reaction mixture is pouredinto 10 kg of ice, 1.5 liters of concentrated hydrochloric acid and 1.5kg of citric acid. 1 kg of Hyflo Super Cel is added, and the mixture isstirred for 1 hour and then filtered; the residue is washed with 2liters of water, 2×2 liters of toluene and, finally, 2×2 liters of 1NHCl solution. The first filtrate and the washing water are combined; theaqueous phase is separated off and extracted 2× with the two toluenefiltrates. The resulting organic phases are washed with the twohydrochloric-acid-containing filtrates. The aqueous phases are combined;6 liters of toluene are added and the mixture is adjusted to a pH offrom 8 to 9 with 4.6 liters of sodium hydroxide solution (30% in water).The mixture is filtered through Hyflo (filtration aid based onkieselguhr, Fluka, Buchs, Switzerland); the aqueous phase is separatedoff and extracted 2× with 2 liters of toluene. The organic phases arewashed 2× with water, dried (Na₂SO₄) and treated with active carbon.Addition of 0.5 kg of silica gel, stirring, filtration and concentrationby evaporation yield the title compound (physical data as Example 37b).

*b)N-1-(tert-Butoxycarbonyl)-N-2-{4-[(pyridin-2-yl)-phenyl]-methylidene}-hydrazone

A solution of 1770 g (9.67 mol) of 4-(pyridin-2-yl)-benzaldehyde and1220 g (9.2 mol) of tert-butyl carbazate (Fluka, Buchs, Switzerland) in12.5 liters of ethanol is heated at boiling for 4 hours. The mixture iscooled to 40° C. and 6 kg of ice are added; the mixture is filtered offand the title compound is washed with 6 liters of water, that compoundthen being obtained in pure form (physical data as in Example 37c).

*c) N-1-(tert-Butoxycarbonyl)-N-2-[4-(pyridin-2-yl)-benzyl]-hydrazine

A suspension of 1655 g (5.57 mol) ofN-1-(tert-butoxycarbonyl)-N-2-{4-[(pyridin-2-yl)-phenyl]-methylidene}-hydrazonein 12 liters of methanol is hydrogenated in the presence of 166 g of 10%Pd/C under normal pressure at room temperature. The catalyst is filteredoff and washed thoroughly with methanol; the solvent is removed.Crystallisation from hexane yields the title compound: m.p.: 74-77° C.

*d)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azahexane

A solution of 1185 g (4.5 mol) of(2R)-[(1′S)-(tert-butoxycarbonyl)-amino-2′-phenylethyl]-oxirane and 1230g (4.1 mol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(pyridin-2-yl)-benzyl]-hydrazine in 14liters of iso-propanol are heated at boiling for 16 hours. Aftercooling, 15 kg of ice and 10 liters of water are added; the mixture isstirred for 2 hours; the crystals are filtered off and washed with 6liters of water. Stirring twice in 5 liters of ether in each case,filtration, washing with 2 liters of ether and, finally, 2 liters ofether/tert-butyl methyl ether 1:1 yield the title compound: m.p.:183-188° C.

*e)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-diamino-6-phenyl-2-azahexanetrihydrochloride

A solution of 1465 g (2.6 mol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[(tert-butoxycarbonyl)amino]-6-phenyl-2-azalbexanein 12 liters of THF and 4 liters of hydrochloric acid (4N in water) isstirred at 50° C. for 4 hours. The aqueous phase is separated from theresulting two-phase mixture and concentrated by evaporation in vacuo.The residue is diluted with 4 liters of ethanol, concentrated byevaporation, diluted with 4 liters of ethanol/toluene 1:1, concentratedby evaporation, diluted with 4 liters of ethanol and concentrated byevaporation again. Stirring in 9 liters of DIPE and filtration yield thetitle compound (physical data as Example 37f).

*e(i): Alternatively,1-(4-(pyridin-2-yl)-phenyl)-4(S)-hydroxy-5(S)-2,5-di[(tert-butoxycarbonyl)-amino]-6-phenyl-2-azahexaneis prepared as follows:

Under a nitrogen atmosphere, 2.1 ml (2.1 mmol) of a 1.00M solution ofdiisobutylaluminium hydride in methylene chloride are slowly addeddropwise to an ice-cooled solution of 200 mg (0.347 mmol) of1-[4-(pyridin-2-yl)phenyl]-1-oxo-5-(S)-2,5-di[(tert-butoxycarbonyl)-amino]-4(S)-hydroxy-6-phenyl-2-azahexanein 5 ml of THF (foams). After 2 hours, 7 ml of ethyl acetate are addedand, after a further 30 min, 70 ml of methanol. The reaction mixture iswarmed to room temperature and stirred for 2 hours; 0.5 ml of water and5 g of sodium sulfate are added and the mixture is stirred again for 1hour to complete the reaction. The salts are filtered off and thefiltrate is concentrated by evaporation. Medium-pressure chromatography(SiO₂, hexane/ethyl acetate 3:2→ethyl acetate) yields the titlecompound: m.p. 184° C.; TLC (hexane/ethyl acetate 1:1): R_(f)=0.26; FABMS (M+H)⁺=563.

The synthesis of the starting material,1-[4-(pyridin-2-yl)phenyl]-1-oxo-5-(S)-2,5-di[(tert-butoxycarbonyl)amino]-4(S)-hydroxy-6-phenyl-2-azahexane,is carried out via the following steps:

Step (1) 4-(Pyridin-2-yl)-benzoic acid methyl ester:

24.0 g (150 mmol) of 4-cyanobenzoic acid methyl ester (Fluka, Buchs,Switzerland) in 150 ml of toluene are placed under an acetyleneatmosphere in an autoclave and 0.30 g (1.6 mmol) of cobaltocene(=dicyclopentadienylcobalt; Aldrich, Milwaukee, USA) is added. Themixture is then subjected to an acetylene pressure of 15 atm, heated at180° C. and stirred for 12 hours. After cooling and release of thepressure, 9.5 g of active carbon are added to the black suspension; themixture is diluted with 250 ml of toluene, stirred for 30 min, filteredand concentrated by evaporation. Crystallisation from warm ether by theaddition of hexane yields the title compound: m.p. 96° C.; TLC(hexane/ethyl acetate 4:1): R_(f)=0.37; FAB MS (M+H)⁺=214. Furtherproduct can be obtained from the mother liquor by column chromatography(SiO₂, hexane/ethyl acetate 19:1→4:1).

Step (2) 4-(Pyridin-2-yl)-benzoic acid:

12.85 g (60.2 mmol) of 4-(pyridin-2-yl)-benzoic acid methyl ester in 125ml of methanol and 67 ml of 1N sodium hydroxide solution are stirred atroom temperature for 6 hours. The resulting solution is partiallyconcentrated by evaporation; the aqueous residue is extracted with ethylacetate and acidified to pH≈1.5 with 2N HCl solution. The title compoundprecipitates out and can be filtered off and washed with water: TLC(ethyl acetate): R_(f)=0.35; FAB MS (M+H)⁺=200.

Step (3) 4-(Pyridin-2-yl)-benzoic acid isobutyloxyformic acid anhydride:

With the exclusion of air, 6.0 g (30 mmol) of 4-(pyridin-2-yl)-benzoicacid are suspended at −20° C. in 90 ml of THF, and 9.90 ml (90 mmol) ofN-methyl-morpholine and 4.32 ml (33 mmol) of isobutyl chloroformate areadded. After 30 min, the mixture is filtered, washed with a small amountof cold THF, and the filtrate is partially concentrated by evaporation;the residue is diluted with methylene chloride, washed with ice-waterand cold brine, dried (Na₂SO₄) and concentrated by evaporation to formthe title compound: ¹H-NMR (CDCl₃) i.a. 8.75 (m, 1H), 8.16 (AB, J=8,4H), 7.81 (m, 2H), 7.32 (4-line system, J=5, 1H), 4.16 (d, J=7, 2H),2.10 (9-line system, J=7, 1H), 1.02 (d, J=7, 6H).

Step (4) 1-(R)-Cyano-2(S)-(N-tert-butoxycarbonylamino)-3-phenylpropyl[4-(2-pyridyl)]-benzoate:

At 0° C., 250 mg (0.9 mmol) of benzyltriethylammonium chloride are addedto 2.0 g (30 mmol) of potassium cyanide in 7.5 ml of water and 7.5 ml ofmethylene chloride. Then a solution of 6.21 g (24.9 mmol) ofBoc-(L)-phenylalaninal in 10 ml of methylene chloride and a solution of≈30 mmol of 4-(pyridin-2-yl-benzoic acid-iso-butyloxyformic acidanhydride in 10 ml of methylene chloride are simultaneously addeddropwise. After 20 min at 0° C., stirring is carried out at roomtemperature for a further 4 hours and the reaction mixture is finallydiluted with methylene chloride/water. The aqueous phase is separatedoff and extracted 2× with methylene chloride; the organic phase iswashed 3× with water and brine, dried (Na₂SO₄) and concentrated byevaporation. Column chromatography (SiO₂; hexane/ethyl acetate 4:1→2:1)yields a ≈5:1 mixture of1-(R)-cyano-2(S)-(N-tert-butoxycarbonylamino)-3-phenylpropyl[4-(2-pyridyl)]-benzoate and1-(S)-cyano-2(S)-(N-tert-butoxycarbonylamino)-3-phenylpropyl[4-(2-pyridyl)]-benzoate: TLC (hexane/ethyl acetate 4:1): R_(f)=0.11;FAB MS (M+H)⁺=458; ¹H-NMR (CDCl₃) i.a. 5.66 (d, J=6, ⅚H, 1-(R) epimer),5.53 (m, ⅙H, 1-(S) epimer). Digestion in DIPE results indiastereoisomerically pure1-(R-cyano-2(S)-(N-tert-butoxycarbonylamino)-3-phenylpropyl[4-(2-pyridyl)]-benzoate: m.p. 140-141° C.

Step (5)4-(S)-1,4-Di[(tert-butoxycarbonyl)amino]-3(R)-[4-(pyridin-2-yl)phenyl]-carbonyloxy-5-phenyl-1-azapent-1-ene:

2.29 g (5.0 mmol) of1-(R-cyano-2(S)-(N-tert-butoxycarbonylamino)-3-phenylpropyl[4-(2-pyridyl)]-benzoate are dissolved in 80 ml of methanol, and 900 mg(15 mmol) of acetic acid and 661.5 mg (5 mmol) of tert-butyl carbazateare added; after the addition of 2.3 g of Raney nickel, the mixture ishydrogenated. The partially precipitated product is dissolved by theaddition of methanol and gentle heating; the catalyst is filtered offand the filtrate is concentrated by evaporation. The residue is taken upin ethyl acetate/sat. NaHCO₃ solution; the aqueous phase is separatedoff and extracted a further 2× with ethyl acetate. The organic phasesare washed with brine, dried (Na₂SO₄) and concentrated by evaporation.Medium-pressure chromatography (SiO₂; hexane/ethyl acetate 4:1→ethylacetate) yields the title compound: m.p. 195-196° C.; TLC (hexane/ethylacetate 1:1): R_(f)=0.39; FAB MS (M+H)⁺=575.

Step (6)1-[4-(Pyridin-2-yl)phenyl]-1-oxo-5-(S)-2,5-di[(tert-butoxycarbonyl)amino]-4(S)-hydroxy-6-phenyl-2-azahexane

Under a nitrogen atmosphere, 111 mg (85%; 1.5 mmol) of NaCNBH₃ are addedto a solution of 862 mg (1.5 mmol) of4-(S)-1,4-di[(tert-butoxycarbonyl)amino]-3(R)-[4-(pyridin-2-yl)phenyl]-carbonyloxy-5-phenyl-1-azapent-1-enein 10 ml of THF. A solution of 290 mg (1.5 mmol) of p-toluenesulfonicacid in 4 ml of THF is added dropwise thereto. After stirring for 2.5hours, a further 55 mg of NaCNBH₃ and 145 mg of p-toluenesulfonic acidin 2 ml of THF are added and the mixture is stirred again for 2.5 hours.The reaction mixture is then poured into 230 ml of a 1% solution ofK₂B₄O₇.4H₂O in water, stirred overnight to complete the reaction,filtered and washed with water. The residue is taken up in ethylacetate; the solution is washed with brine, dried (Na₂SO₄) andconcentrated by evaporation{→4-(S)-1,4-di[(tert-butoxycarbonyl)amino]-3(S)-[4-(pyridin-2-yl)phenyl]-carbonyloxy-5-phenyl-1-azapentane:TLC (hexane/ethyl acetate 1:1): R_(f)=0.45}. The resulting foam isdissolved in 25 ml of diethylene glycol dimethyl ether; 250 μl of7-methyl-1,5,7-triaza-bicyclo[4.4.0]dec-5-ene (Fluka; Buchs,Switzerland) are added and the mixture is heated at 80° C. for 1.5hours. The mixture is concentrated by evaporation under a high vacuumand the residue is taken up in ethyl acetate/water; the aqueous phase isseparated off and extracted a further 2× with ethyl acetate. The organicphases are washed with brine, dried (Na₂SO₄) and concentrated byevaporation. Crystallisation from DIPE/hexane yields the title compound:m.p. 104-105° C.; TLC (hexane/ethyl acetate 1:1): R_(f)=0.20; FAB MS(M+H)⁺=577.

Example 47[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-iso-leucyl)amino]-phenyl-2-azahexane

Under a nitrogen atmosphere, 0.45 g (1.5 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 0.85 g (4.5 mmol) of EDC and 0.4 g (3mmol) of HOBT are dissolved in 10 ml of DMF. After the addition of 1.26ml of TEA and stirring for 10 min, a solution of 0.96 g (1.5 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbony-(L)-iso-leucyl)-amino-6-phenyl-2-azahexanehydrochloride in 10 ml of DMF is then added dropwise. After 2 hours, thereaction mixture is concentrated by evaporation. The resulting oil istaken up in methylene chloride and washed with water, 2× sat. NaHCO₃solution, water and brine. The aqueous phases are extracted withmethylene chloride; the combined organic phases are dried (Na₂SO₄) andconcentrated by evaporation. The residue is digested first in DIPE andthen in methylene chloride/ether, then filtered off and dried to yieldthe title compound: TLC: R_(f)=0.45 (ethyl acetate); HPLC₂₀₋₁₀₀:t_(Ret)=11.71; FAB MS (M+H)⁺=705.

The starting material is prepared as follows:

47a)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-(N-Boc-amino)-5(S)-trifluoroacetyl-amino-6-phenyl-2-azahexane

Analogously to Example 37e), 7 g (23 mmol) ofN-1-(tert-butoxycarbonyl)-N-2-[4-(pyridin-2-yl)-benzyl]-hydrazine arereacted with 6 g (23 mmol) of(2(R)-[(1′S)-trifluoroacetyl-amino-2′-phenylethyl]oxirane in 125 ml ofisopropanol at 80° C. to form the title compound. TLC: R_(f)=0.33(methylene chloride/methanol: 1/1); HPLC₂₀₋₁₀₀: t_(Ret)=12.76; FAB MS(M+H)⁺=559.

47b)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-(N-Boc-amino)-5(S)-amino-6-phenyl-2-azahexane

Analogously to Example 40e, 5.6 g (10 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2-(N-Boc-amino)-5-(trifluoroacetyl-amino)-6-phenyl-2-azahexaneare dissolved in 130 ml of methanol, heated to 65° C. and converted intothe title compound by the dropwise addition of 50 ml of a 1M aqueouspotassium carbonate solution. TLC: R_(f)=0.17 (methylenechloride/methanol: 9/1); HPLC₂₀₋₁₀₀: t_(Ret)=8.50; FAB MS (M+H)⁺=463.

47c)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-Boc-amino-5(S)-N-(N-methoxy-carbonyl-(L)-isoeucyl)amino-6-phenyl-2-azahexane

Analogously to Example 1, a solution of 1.62 g (3.5 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-(N-Boc-amino)-5(S)-amino-6-phenyl-2-azahexanein 25 ml of DMF is added dropwise to a mixture of 1.06 g (5.6 mmol) ofN-methoxycarbonyl-(L)-iso-leucine, 2.01 g (10.5 mmol) of EDC and 0.95 g(7 mmol) of HOBT in 20 ml of DMF. After working up, the crude product isdigested in DIPE, filtered off and dried. TLC: R_(f)=0.59 (ethylacetate); HPLC₂₀₋₁₀₀: t_(Ret)=12.52. FAB MS (M+H)⁺=634.

47d)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexanehydrochloride

Analogously to Example 40g, 40 ml of 4M HCl in dioxane are added to 1.9g (3 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-Boc-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexaneand the mixture is diluted with 3 ml of DMF. After 2.5 hours, themixture is worked up. The title compound is obtained: TLC: R_(f)=0.55(methylene chloride/methanol: 9/1); HPLC₂₀₋₁₀₀: t_(Ret)=8.74; FAB MS(M+H)⁺=534.

Example 481-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxy-carbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexane

Analogously to Example 1, a solution of 0.964 g (1.5 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-tert-leucyl)amino-6-phenyl-2-azahexanehydrochloride in 10 ml of DMF is added dropwise to a mixture of 0.42 g(2.4 mmol) of N-methoxycarbonyl-(L)-valine, 0.862 g (4.5 mmol) of EDC,0.405 g (3 mmol) of HOBT and 1.26 ml of TEA in 10 ml of DMF. Afterworking up, the crude product is digested in DIPE, filtered off anddried. Subsequent column chromatography (SiO₂; hexane/ethyl acetate: 1/1to 3/1) yields the pure title compound (TLC: R_(f)=0.35 (ethyl acetate);HPLC₂₀₋₁₀₀: t_(Ret)=10.9. FAB MS (M+H)⁺=691.

Example 491-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxy-carbonyl-(L)-tert-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Analogously to Example 1, a solution of 0.315 g (0.5 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanehydrochloride in 3 ml of DMF is added dropwise to a mixture of 0.152 g(0.8 mmol) of N-methoxycarbonyl-(L)-tert-leucine, 0.287 g (1.5 mmol) ofEDC, 0.135 g (1 mmol) of HOBT and 0.49 ml of TEA in 3 ml of DMF. Afterworking up, the crude product is purified by subsequent medium-pressurecolumn chromatography (SiO₂; hexane/ethyl acetate) to yield the titlecompound. TLC: R_(f)=0.35 (ethyl acetate); HPLC₂₀₋₁₀₀: t_(Ret)=11.05.FAB MS (M+H)⁺=691.

The starting compounds are prepared as follows:

49a)1-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-Boc-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Analogously to Example 1, a solution of 4.1 g (8.87 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-(N-Boc-amino)-5(S)-amino-6-phenyl-2-azahexane(Example 47 b) in 50 ml of DMF is added dropwise to a mixture of 2.49 g(14.2 mmol) of N-methoxycarbonyl-(L)-valine, 5.1 g (26.6 mmol) of EDC,2.4 g (17.7 mmol) of HOBT and 7.45 ml of TEA in 50 ml of DMF. Afterworking up, the crude product is digested 2× in DIPE, filtered off anddried to yield the title compound. TLC: R_(f)=0.42 (ethyl acetate);HPLC₂₀₋₁₀₀: t_(Ret)=11.92. FAB MS (M+H)⁺=620.

49b)1-[4(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanehydrochloride

Analogously to Example 37f), 30 ml of 4M HCl in dioxane are added to 3.5g (5.65 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-Boc-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexaneand the mixture is diluted with 5 ml of DMF. After 3.5 hours, themixture is worked up. The title compound is obtained: TLC: R_(f)=0.53(methylene chloride/methanol: 9/1); HPLC₂₀₋₁₀₀: t_(Ret)=8.00; FAB MS(M+H)⁺=520.

Example 501-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxy-carbonyl-(L)-valyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane

Analogously to Example 46, 0.96 g (1.5 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-iso-leucyl)amino-6-phenyl-2-azahexane3HCl (Example 47d) in 10 ml of DMF are reacted with 0.263 g (1.5 mmol)of N-methoxycarbonyl-(L)-valine, 0.446 g (1.5 mmol) of TPTU and 0.78 ml(4.5 mmol) of DBU in 7 ml of DMF. After working up, the title compoundis obtained: TLC: R_(f)=0.4 (ethyl acetate); HPLC₂₀₋₁₀₀: t_(Ret)=11.23.FAB MS (M+H)⁺=691.

Example 511-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-methoxy-carbonyl-(L)-iso-leucyl)amino-5(S)-N-(N-methoxycarbonyl-(L)-valyleamino-6-phenyl-2-azahexane

Analogously to Example 1, a solution of 1.26 g (2 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexanehydrochloride (Example 49b) in 12 ml of DMF is added dropwise to amixture of 0.6 g (3.2 mmol) of N-methoxycarbonyl-(L)-iso-leucine, 1.14 g(6 mmol) of EDC, 0.54 g (4 mmol) of HOBT and 1.68 ml of TEA in 13 ml ofDMF. After working up, the crude product is digested in DIPE andpurified by subsequent medium-pressure column chromatography (SiO₂;hexane/ethyl acetate) to yield the title compound. TLC: R_(f)=0.32(ethyl acetate); HPLC₂₀₋₁₀₀: t_(Ret)=11.04. FAB MS (M+H)⁺=691.

Example 521-[4-(Pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-N-(N-ethoxy-carbonyl-(L)-valyl)-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyl)amino-6-phenyl-2-azahexane

Analogously to Example 1, a solution of 0.629 g (1 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-2-amino-5(S)-N-(N-methoxycarbonyl-(L)-valyleamino-6-phenyl-2-azahexanehydrochloride (Example 49b) in 5 ml of DMF is added dropwise to amixture of 0.303 g (1.6 mmol) of N-ethoxycarbcnyl-(L)-valine, 0.575 g (3mmol) of EDC, 0.27 g (2 mmol) of HOBT and 0.98 ml of TEA in 7 ml of DMF.After working up, the crude product is digested in DIPE and purified bysubsequent medium-pressure column chromatography (SiO₂; hexane/ethylacetate) to yield the title compound. TLC: R_(f)=0.33 (ethyl acetate);HPLC₂₀₋₁₀₀: t_(Ret)=11.13. FAB MS (M+H)⁺=691.

Example 531-[4-(Pyrid-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexanemethanesulfonate salt

210 mg (0.28 mmol) of1-[4-(pyridin-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane(Example 46) are dissolved in 10 ml of methylene chloride with heatingand 19.5 μl (0.3 mmol) of methanesulfonic acid are added. The titlecompound is precipitated with ether, filtered off and dried underreduced pressure at 50° C. FAB MS (M+H)⁺=705. ¹H-NMR (CD₃OD) (chemicalshifts of the pyridine protons of the free base in brackets); δ: 8.81(8.6), 8.65 (7.9), 8.36 (7.8), 8.05 (7.35) and also, in addition,signals of the methyl group of the salt: δ: 2.7 ppm.

Example 541-[4-(Pyrid-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexanehydrochloride salt

70 mg (0.094 mmol) of1-[4-(pyrid-2-yl)-phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxy-carbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane(Example 46) are dissolved in 6 ml of dioxane, and 25 μl of a 4M HClsolution in dioxane are added. The resulting precipitate is filtered offand dried. FAB MS (M+H)⁺=705. ¹H-NMR (CD₃OD) (chemical shifts of thepyridine protons of the free base in brackets); δ: 8.81 (8.6), 8.65(7.9), 8.36 (7.8), 8.05 (7.35). Elemental analysis of the hydrate of thetitle compound: Cl found: 4.6%; calc.: 4.63%.

Example 55: Gelatin Solution

A sterile-filtered aqueous solution, containing 20% cyclodextrins assolubiliser, of one of the compounds of formula I mentioned in thepreceding Examples (e.g. the title compound from Example 2) as activeingredient, is so mixed, with heating and under aseptic conditions, witha sterile gelatin solution containing phenol as preservative that 1.0 mlof solution has the following composition:

active ingredient 3 mg gelatin 150 mg phenol 4.7 mg dist. watercontaining 20% cyclodextrins 1.0 ml as solubiliser

Example 56: Sterile Dry Substance for Injection

5 mg of one of the compounds of formula I mentioned in the precedingExamples (for example the title compound from Example 3) as activeingredient are dissolved in 1 ml of an aqueous solution containing 20 mgof mannitol and 20% cyclodextrins as solubiliser. The solution issterile-filtered and, under aseptic conditions, introduced into a 2 mlampoule, deep-frozen and lyophilised. Before use, the lyophilisate isdissolved in 1 ml of distilled water or 1 ml of physiological saline.The solution is administered intramuscularly or intravenously. Theformulation can also be introduced into double-chamber disposablesyringes.

Example 57: Nasal Spray

500 mg of finely ground (<5.0 mm) powder of one of the compounds offormula I mentioned in the preceding Examples (for example the compoundfrom Example 4) are suspended as active ingredient in a mixture of 3.5ml of Myglyol 812® and 0.08 g of benzyl alcohol. The suspension isintroduced into a container having a metering valve. 5.0 g of Freon 12®(dichlorodifluoromethane; trade mark of DuPont) are introduced underpressure through the valve into the container. The “Freon” is dissolvedin the Myglyol/benzyl alcohol mixture by shaking. The spray containercontains approximately 100 single doses which can be administeredindividually.

Example 58: Film-coated Tablets

The following constituents are processed for the preparation of 10 000tablets each comprising 100 mg of active ingredient:

active ingredient 1000 g corn starch  680 g colloidal silicic acid  200g magnesium stearate  20 g stearic acid  50 g sodium carboxymethylstarch  250 g water quantum satis

A mixture of one of the compounds of formula I mentioned in thepreceding Examples (for example the compound from Example 5) as activeingredient, 50 g of corn starch and the colloidal silicic acid isprocessed with a starch paste made from 250 g of corn starch and 2.2 kgof demineralised water to form a moist mass. That mass is forced througha sieve of 3 mm mesh size and dried in a fluidised bed dryer at 45° for30 min. The dried granules are pressed through a sieve of 1 mm meshsize, mixed with a previously sieved mixture (1 mm sieve) of 330 g ofcorn starch, the magnesium stearate, the stearic acid and the sodiumcarboxymethyl starch and compressed to form slightly convex tablets.

Example 59: Capsules (I)

A compound from one of the afore-mentioned Examples (e.g. the titlecompound from Example 6) is micronised (particle size about 1 to 100 μm)using a customary knife mixer (e.g. Turmix). ®Pluronic F 68 (blockpolymer of polyethylene and polypropylene glycols; Wyandotte Chem.Corp., Michigan, USA; also obtainable from Emkalyx, France; trade markof BASF) is likewise micronised using a customary mixer and the finescontent is removed using a sieve (0.5 mm) and used further as below.16.00 g of sesame oil are placed in a glass beaker and 1.20 g of themicronised active ingredient, 1.20 g of the fines content of ®Pluronic F68 and 1.20 g of hydroxypropylmethylcellulose (Cellulose HP-M-603 fromShin-Etsu Chemicals Ltd., Tokyo, JP) are added with stirring using astirring device (IKA-Werk, FRG) combined with a toothed stirrer(diameter: 46 mm) (stirring speed: 2000 rev/min). Twenty minutes'stirring at the speed indicated produces a suspension of pastyconsistency which is introduced into hard gelatin capsules (20×40 mm; R.P. Scherer AG, Eberbach, FRG).

Example 60: Capsules (II)

For the preparation of 10 000 capsules comprising 100 mg of activeingredient (from one of the afore-mentioned Examples, for example thetitle compound from Example 7) per capsule, the following constituentsare processed as follows:

active ingredient 1000 g ® Pluronic F 68 1000 ghydroxypropylmethylcellulose 1000 g sesame oil 1000 g (for origin ofconstituents see Example 10)

The sesame oil is placed in a heatable vessel (Fryma) and the ®PluronicF 68 is scattered in. The vessel is heated at 60° C. and the ®Pluronic F68 is distributed with stirring (duration about 2 hours). With stirringand homogenisation, the mixture is cooled to about 30° C. Thehydroxypropylmethylcellulose and the active ingredient are scattered inand, with stirring and homogenisation (about 1 hour), distributed in theoil mass. The suspension of pasty consistancy is introduced into hardgelatin capsules (size 0; obtainable, for example, from Elanco orParke-Davies (Caprogel)) or soft gelatin capsules (20 mm oblong; R. P.Scherer AG, Eberbach, FRG) using customary apparatus.

Example 61: Dispersion

For the preparation of a dispersion comprising 120.0 mg of activeingredient 0 ml (preferably the title compound from Example 46), thefollowing constituents are processed as follows:

active ingredient 120.0 mg ® Klucel HF (hydroxypropylcellulose;Hercules, Germany) 50.0 mg ® Tween 20 (polyoxyethylene sorbitanmonolaurate; 100.0 mg Fluka, Buchs, Switzerland) demineralised water10.0 ml

The demineralised water is placed in a container; thehydroxypropylcellulose is scattered in slowly with stirring using amagnetic stirrer and allowed to swell for 1 hour. The polyoxyethylenesorbitan monolaurate is then added and the mixture is stirred for 5 minusing the magnetic stirrer. Finally, the active ingredient is added andthe mixture is stirred for 15 min using the magnetic stirrer.

Example 62: Inhibitory Activity in Respect of HIV-1-protease

Using the system described above with the icosapeptideRRSNQVSQNYPIVQNIQGRR, the IC₅₀ values given below are obtained for thefollowing Examples:

Example IC₅₀ (μM)  1 0.032  2 0.014  3 0.041  4 0.038  5 0.04  6 0.022 7 0.013  8 0.01  9 0.019 10 0.02 11 0.037 12 0.02 13 0.032 14 0.031 150.05 16 0.033 17 0.018 18 0.025 19 0.022 20 0.015 21 0.043 22 0.04 230.034 24 0.05 25 0.1 26 0.021 27 0.027 27 0.051 (1-methyl-1H- tetrazolylisomer) 28 0.083 29 0.014 30 0.054 31 0.171 34 0.072 35 0.058 37 0.02938 0.085 39 0.012 40 0.021 41 0.032 42 0.015 43 0.037 44 0.029 45 0.01246 0.026 47 0.04 48 0.031 49 0.02 50 0.028 51 0.034 52 0.034

Example 63: Protection of MT-2 Cells Against HIV Infection

Using the afore-mentioned test system, in inhibiting the infection ofMT-2 cells by the virus strain HIV-1/MN the title compound from Example46,1-[4-(pyridin-2-yl)pheny]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane,has the following ED₉₀ value: ED₉₀=0.003 μM.

Example 64: Blood Levels in Mice

Using the afore-mentioned test system for the determination of thepharmacokinetics of compounds of formula I, the title compound fromExample 46,1-[4-(pyridin-2-yl)phenyl]-4(S)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyleamino]-6-phenyl-2-azahexane,exibits in mice the following blood levels after oral administration of120 mg/kg:

after administration 30 min 90 min 21.83 31.76

Example 65: Formulation as Solution (I)

The formulation comprises 100 mg of the title compound from Example 46as active ingredient, 100 mg of racemic lactic acid (90%),Cellulose-HP-M-603, silica gel (Aerosil 200) and deionised water (2 g).

Example 66: Formulation as Solution (II)

The formulation comprises 18.4 mg of the title compound from Example 46as active ingredient, 5 mg of Cellulose-HPM-603, 40 mg ofN-methylpyrrolidone and double-distilled water ad 1 ml.

Example 67: Analogously to One of the Afore-mentioned Processes, Thereare Prepared

A)1-[4-(pyridin-2-yl)phenyl]-4(R)-hydroxy-5(S)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane;

B)1-[4-(pyridin-2-yl)phenyl]-4(R)-hydroxy-5(R)-2,5-bis[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane;

C)1-[4-(pyridin-2-yl)phenyl]-4(S)-hydroxy-5(S)-2-[N-(N-methoxycarbonyl-(L)-tert-leucyl)-amino]-5-[N-(N-methoxycarbonyl-(D)-tert-leucyl)amino]-6-phenyl-2-azahexane;or

D)1-[4-(pyridin-2-yl)phenyl]-4(S)-hydroxy-5(S)-2-[N-(N-methoxycarbonyl-(D)-tert-leucyl)-amino]-5-[N-(N-methoxycarbonyl-(L)-tert-leucyl)amino]-6-phenyl-2-azahexane.

What is claimed is:
 1. A process for the preparation of a compound ofthe following formula I*,

wherein R₁ is lower alkoxycarbonyl, R₂ is secondary or tertiary loweralkyl or lower alkylthio-lower alkyl, R₃ is phenyl that is unsubstitutedor substituted by one or more lower alkoxy radicals, or C₄-C₈cycloalkyl,R₄ is phenyl or cyclohexyl each substituted in the 4-position byunsaturated heterocyclyl that is bonded by way of a ring carbon atom,has from 5 to 8 ring atoms, contains from 1 to 4 hetero atoms selectedfrom nitrogen, oxygen, sulfur, sulfinyl and sulfonyl and isunsubstituted or substituted by lower alkyl or by phenyl-lower alkyl,R₅, independently of R₂, has one of the meanings mentioned for R₂, andR₆, independently of R₁, is lower alkoxycarbonyl, or a salt thereof,provided that at least one salt-forming group is present; wherein a) ahydrazine derivative of formula I*

wherein the radicals R₄, R₅ and R₆ are as defined for compounds offormula I, is added to an epoxide of formula IV*,

wherein the radicals R₁, R₂ and R₃ are as defined for compounds offormula I*, free functional groups selected from carboxy, hydroxy oramino group with the exception of those participating in the reactionbeing, if necessary, in protected form, and any protecting groups areremoved, or b) an amino compound of formula V*,

wherein the radicals R₁, R₂, R₃ and R₄ are as defined for compounds offormula I*, is condensed with an acid of formula

or with a reactive acid derivative thereof, wherein the radicals R₅ andR₆ are as defined for compounds of formula I*, free functional groupsselected from carboxy, hydroxy or amino group with the exception ofthose participating in the reaction being, if necessary, in protectedform, and any protecting groups are removed, or c) an amino compound offormula VII*,

wherein the radicals R₃, R₄, R₅ and R₆ are as defined for compounds offormula I*, is condensed with an acid of formula I*

or with a reactive acid derivative thereof, wherein R₁ and R₂ are asdefined for compounds of formula I*, free functional groups selectedfrom carboxy, hydroxy or amino group with the exception of thoseparticipating in the reaction being, if necessary, in protected form,and any protecting groups are removed, or d) to prepare a compound offormula I* wherein the pairs of substituents R₁ and R₆ and R₂ and R₅ arein each case two identical radicals, as defined for compounds of formulaI*, and R₃ and R₄ are as defined for compounds of formula I*, a diaminocompound of formula IX*

wherein the radicals are as just defined, is condensed with an acid offormula I*

or with a reactive acid derivative thereof, wherein R₁′ and R₂′ are asdefined for R₁ and R₆ and for R₂ and R₅, respectively, in formula I*,the pairs R₁ and R₆ and R₂ and R₅ being in each case two identicalradicals, free functional groups with the exception of thoseparticipating in the reaction being, if necessary, in protected form,and any protecting groups selected from carboxy, hydroxy or amino groupare removed, or e) an imino compound of formula (I′)*,

wherein the radicals R₁, R₂, R₃, R₅ and R₆ are as defined for compoundsof formula I*, is reacted with a compound of formula X,

wherein X is a leaving group and R₄ is as defined for compounds offormula I*, free functional groups selected from carboxy, hydroxy oramino group with the exception of those participating in the reactionbeing, if necessary, in protected form, and any protecting groups areremoved, or f) an imino compound of formula (I′)*,

wherein the radicals R₁, R₂, R₃, R₅, and R₆ are as defined for compoundsof formula I*, is reacted with an aldehyde of formula X*,

wherein R₄ is as defined for compounds of formula I*, or with a reactivederivative thereof, with reductive alkylation, free functional groupswith the exception of those participating in the reaction being, ifnecessary, in protected form, and any protecting groups are removed,and, a compound of formula I* having at least one salt-forming groupobtainable in accordance with any one of processes a) to f) above isconverted into its salt or an obtainable salt is converted into the freecompound or into a different salt and/or isomeric mixtures which may beobtainable are separated and/or a compound of formula I* according tothe invention is converted into a different compound of formula I*according to the invention.